FI65447C - FOERFARANDE FOER FRAMSTAELLNING AV EN BAKTERIE SOM INNEHAOLLEREN FOER TILLVAEXTHORMON KODANDE NUCLEOTIDSEKVENS - Google Patents

Description

65447

A method for producing a bacterium containing a nucleotide sequence encoding growth hormone

This invention relates to a method for producing a bacterium containing a nucleotide sequence encoding growth hormone.

The method according to the invention is characterized in that a) the mRNA is isolated from the pituitary cells by homogenization in the presence of a ribonuclease inhibitor, for example about 4 mol / l guanidinium thiocyanate and 0.05 ... 1.0 mol / l / m a solution containing captoethanol at a pH of 5.0 to 8.0, so that no ribonuclease degradation of the mRNA occurs, b) a cDNA having a growth hormone coding 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 is the same enzyme as in d) below, after which the cDNA is cleaved by said restriction endonuclease opening the bacterial plasmid vector with a restriction endonuclease, e.g. in particular by means of Hind III, Hsu I or Eco R1, in a manner known per se, for example by incubation at pH 7.6 at 37 [deg.] C. for 2 hours in order to obtain an open plasmid vector with cohesive ends, e) hydrolysing the 5'-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 35, whereby the cohesive ends mentioned in d) cannot be joined together, 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, for example at pH 7.6 at 14 ° C for one hour, using a molar excess of the plasmid vector , and g) mixing a bacterium such as E. coli, for example E. coli X-1776, and the reaction product obtained in f) to obtain a bacterium containing a nucleotide-10 sequence encoding growth hormone.

The following symbols and abbreviations are used in this specification: DNA - deoxyribonucleic acid 15 RNA - ribonucleic acid

cDNA - complementary DNA

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

20 dATP - deoxyadenosine triphosphate dTTP - deoxythymidine triphosphate dGTP - deoxyguanosine triphosphate dCTP - deoxycytidine triphosphate A - adenine 25 T - thyrine G - guanine C - cytosine

Tris - 2-amino-2-hydroxyethyl-1,3-propanediol EDTA - ethylenediaminetetraacetic acid 30 ATP - adenosine triphosphate TTP - thymidine triphosphate 65447 l

Hind III recognition site --sekvenssi A VAGCTT, specifically a broken arrow into the Hind III endonuclease using Hsu I recognition site - A sequence lAGCTT, specifically a broken arrow mark Hsu I endonuclease means.

5 HaeIII recognition site - SEQ ID GG Ice specifically cleaved by the arrow HaeIII endonuclease means.

Col E1 - Colicin E1-forming plasmid poly dA - polydeoxyadenylate 10 oligo ^ 12-18 "oligodeoxythymidylate (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 the direction of arrow 15 cleaved with Bam HI - endonucleases.

BglII-recognition site - SEQ ID GCCNNNN J'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 20 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 RII endonuclease.

Search II recognition site - SEQ ID AGCGcIt, AGCGci-C, 25 GGCGC or GGCGclc, specifically cleaved by the arrow II endonuclease get through.

Hha I recognition site - restriction endonuclease (obtained from Haemophilus aegyptious microorganism Hinc II recognition site - sequence GTTAAC, GTTGAC, 30 GTCAAG or GTCGAC, specifically cleaved by Hinc II endonuclease.

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

Sal I recognition site - SEQ G ^ TCGAC, speci-35 chromatography cleaved the arrow into the Sal I endonucleases.

4 65447

The Sst I - the recognition site - SEQ ID GAGCT C ^, specifically a broken arrow into the Sst I endonuclease 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 replicates 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 20 triplet sequences corresponding to the amino acid sequence of the protein. The genetic code is shown in the following table.

Genetic code

Phenylalanine (Phe) RTD Histidine (His) CAK

Leucine (Leu) XTY Glutamine (Gin) CAJ

25 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

30 Threonine (Thr) ACL Tryptophan (Try) TGG

Alanine (Lower) GCL Arginine (Arg) WGZ

Tyrosine (Tyr) TAK Glycine (Gly) GGL

Terminal signal TAJ

Terminal signal TGA

35 Key: Each three-letter triplet represents a DNA trinucleotide with a 5 'end on the left and a 3' end on 65447 right; the letters represent the purine and pyrimidine bases from which the nucleotide sequence is formed.

A = adenine G * guanine 5 C = cytosine T = thyrine

X = T or C if Y is A or G X = G if Y is C or T Y = A, G, C or T if X is C 10 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 15 QR = TC if S is A, G, C or T

QR = AG if S is T or C S * A, G, C or T if QR is TC S = T or C if QR is AG J = A or G 2 0 K = T or C

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

Transcription is the first step in the biological process by which nucleotide sequence information is transformed into 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 uracil is used instead of thymine. 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 inherently messenger RNA (rnRNA) 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 15 Cks. 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 Cks of proinsulin. Cahn., S.J., 20 Keim, P. and Steiner D.F., Proc. Natl. Acad. Sci. USA, 73, 1961 * (1976) and Lomedico, P.T. and Saunders, G.F., Nucl.

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

25 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 industrially, medically or scientifically significant hormones. These proteins are often difficult to isolate in useful amounts, and this problem is quite difficult for human-derived proteins. 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-5 line constant so that the desired specific properties are maintained.

In contrast, microorganisms, such as bacteria, are relatively easy to cultivate in chemically defined media. Fermentation technology is advanced. Cultivation of organisms quickly and in high yields is possible. In addition, some microorganisms have been extensively 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 synthesize 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 synthesized by such a method. In addition, the ability to isolate and transfer the genetic sequence that defines the production of a particular protein into a microorganism with a well-known genetic background 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 can also be transformed to provide proteins with altered therapeutic or functional properties.

The process of this invention is a multi-step process involving enzyme-catalyzed reactions. The nature of these enzyme reactions is described on the basis of 30 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 non-covalent binding of 35 DNA templates to the 3 'end of the RNA, followed by gradual insertion of the required deoxynucleotides into the 3' end of the growing chain as base pairs defined by the mRNA nucleotide sequence δ 65447. 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. The 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, 10 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 15 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 endonucle-20 asases have been isolated from a variety of sources and characterized for recognition targets based on the nucleotide sequence.

Some restriction endonucleases hydrolyze phosphodiester bonds from the same site in each strand to form a blunt end. Some catalyze the hydrolysis of bonds separated by a few nucleotides to form 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 DNA molecules with the same recognition site, the same cohesive ends are generated, and thus it is possible to combine restriction endonuclease-treated heterologous DNA sequences with other similarly treated sequences, cf.

35 Roberts, R.J. Crit. Rev. Biochem. 4, 123 (1978). However, restriction sites are quite rare, but the utility of restriction endonucleases has been enhanced by synthesizing double-stranded oligonucleotides with a recognition site sequence. . Thus, almost any DNA segment can be ligated to any other segment by attaching the required restriction oligonucleotide to the ends of the molecule, exposing the product 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. 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 may be composed 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-25 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 (see Sgaramella, V., Van de Sande, JH and Khorana, HG, Proc. Nat 1. Acad. Sci. USA 67 , 1468 (1970)).

Alkaline phosphatase is a common enzyme capable of hydrolyzing phosphate esters, including the 51-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. Γ] asm idi is the term used for any independently replicating DNA unit that is present in a 10 65447 • raicrobial cell and does not belong to the host cell's own genome. The plasmid is not genetically bound to the chromosome of the host cell. Plasmid DNA exists as circular double-stranded molecules, usually with a molecular weight of a few 8 to 5 million, some even more than 10, and usually represent only a small percentage of the total DNA in 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 proliferation of the host cell, and sometimes the rate of proliferation can be regulated by altering growth conditions. Although the plasmid exists as a closed ring, a DNA segment can be artificially inserted to form a higher molecular weight recombinant plasmid whose essential ability to replicate and express genes has not been substantially altered. 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 suitable for selection purposes, such as genes that confer drug resistance.

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As examples of the specific DNA sequences of this invention, the structural gene for rat growth hormone and human growth hormone is transferred to a Dactor. Thus, it is contemplated that the method may well be applied to the transfer of any DNA sequence taken from a higher organism, such as a vertebrate, to a bacterial host. In this context, a higher organism is defined as any eukaryotic organism containing differentiated tissues, including e.g. insects, molluscs, plants and vertebrates, 10 including mammals, which in turn include cattle, pigs, primates and humans.

In that process, the desired cell is first isolated by an improved method. mRNA is isolated from cells unchanged by a new method in which RNAse-15 activity is virtually eliminated. Intact mRNA is purified from the extract by column chromatography and exposed to a reverse transcriptase enzyme in the presence of the four deoxynucleoside triphosphates required to synthesize the complementary strand (cDNA). In this first step, the product obtained by reverse transcriptase is subjected to a procedure for selectively removing the ribonucleotide sequence. The remaining deoxynucleotide sequence complementary to the original mRNA is incubated in a second reaction with reverse transcriptase and not DNA polymerase in the presence of four deoxynucleoside triphosphates. The product obtained is a double cDNA with complementary strands joined together by a single strand at one end. Thus, this product is treated with a nuclease specific for the single 12 65447 strand, which cleaves the simple connecting strand. The resulting double-stranded cDNA is extended by adding to each end a specific DNA containing the restriction enzyme recognition site sequence.

The addition is catalyzed by DNA-1 igase. The extended cDNA is then treated with a restriction endonuclease that produces self-complementing single-stranded ends at the 5 'end of each strand of the double-strand.

Plasmid DNA having the same restriction endonuclease recognition site is treated with an enzyme to cleave the polynucleotide strand to form self-complementing single-stranded nucleotide sequences at the 5 'ends. The 5 'terminal phosphate groups of the single-stranded ends are removed to prevent the plasmid from forming a ring structure that transforms the host cell. The prepared cDNA and plasmid DNA are incubated together in the presence of DNA ligase. Under the reaction conditions described, viable closed-loop plasmid DNA can only be generated if it contains a cDNA segment. The plasmid containing the cDNA sequence is then transferred to a suitable host cell. Cells that have received a viable plasmid are identified in culture from colonies that have the genetic trait associated with the plasmid, such as drug resistance. Pure bacterial strains with a recombinant plasmid containing the cDNA-25 sequence are then cultured and the recombinant plasmid is re-isolated. In this way, large amounts of recombinant plasmid DNA can be prepared and the specific cDNA sequence can be isolated therefrom by endonucleolytic cleavage using a suitable restriction enzyme.

This invention relates to 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 found after replication in the organism, and the various steps of the method can be divided into four groups: Isolation of the desired cell from a higher organism

There are two possible sources for the genetic code of a particular protein, namely DNA from the source organism or 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 15 have been thoroughly purified or when a particularly high-risk gene is used. working spaces (P4), see Federal Register, Vol. 41, no. 131, 1967-07-07, ss. 27902-27943. Thus, any method for producing a human protein, such as the method of the present invention, must proceed from the isolation of a specific mRNA that contains the code for 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 take advantage of the fact that highly specialized organisms, such as vertebrates, have certain cells at certain sites whose function is to produce 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.

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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-5 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 the separation of cells from connective tissue and the separation of the desired cell type from 10 other types of tissue cells.

1 It has often been found that the proportion of the desired mRNA can be increased by taking advantage of the cell's ability to respond to external stimuli. For example, hormone treatment may cause an increase in the production of the desired mRNA. Other methods include growth at a certain temperature and / or in a particular nutrient or other chemical substance.

2. Extraction of mRNA An important feature of the present invention is that RNase activity is virtually eliminated 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 incapable of transferring the intact genetic sequence to the microorganism.

25 As mentioned above, the RN donkey is widespread and 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 great, as pancreas 30 produces digestive enzymes and is thus very RN-ass. However, the RNase impurity applies to all tissues and the method of destroying RNase activity described herein can be applied to all cells.

65447 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 mRNA.

The choice of suitable chaotropic ions depends on their water solubility and availability. Suitable chaotropic cations include e.g. guanidinium, carbamoylguanidinium, guanylguanidinium, lithium, etc. Suitable chaotropic an-10 ions include e.g. iodide, perchlorate, thiocyanate, diiodo-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 it has a solubility of only about 0.1 M and is also relatively expensive. Guanidinium thiocyanate is the preferred cationic anion combination because it is readily available and has good solubility in aqueous solutions, up to about 5 mol / L.

Thiol compounds, such as β-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 di-thiothreitol, cysteine, propanol dimercaptan, etc. Water solubility is a necessary requirement, as a large excess of thiol compound 25 must be present compared to intramolecular disulfides in order for the exchange reaction to be practically must be given priority as it is easily available at a reasonable price.

30 The efficacy 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 practicable concentration. The success of the present invention in keeping the roRNA 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 a protein often depends on the ratio of the concentration of denaturant to a threshold value of 5 to 10 raised to the power, cf. Tanford, C. A., Adv. Prot. Chem. 23, 121 (1968). Qualitatively, this ratio means that only a slightly more efficient denaturant than guanidinium-10 hydrochloride is able to denature the protein many times faster at the same concentration. The relationship between the kinetics of RNase denaturation and the retention of mRNA during extraction from cells has apparently not been observed or studied prior to this invention.

If the above analysis is correct, the preferred denaturant should be one with a low threshold concentration in denaturation and high water solubility. Therefore, guanidinium thiocyanate is more preferred than lithium diiodic salicylate, although the latter is a more potent denaturant because guanidinium thiocyanate is more soluble and thus can be used at such a concentration that RNase is inactivated more rapidly. The above analysis also explains why guanidinium thiocyanate is more preferred than nearly equally soluble hydrochloride (cyanate is a slightly stronger denaturant than the hydrochloride).

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. The thiol compound is thought to aid in the progress of the denaturation process because it prevents 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 bond cleavage reagents with thiol groups are somewhat effective at any concentration, but it is preferred to use a large excess of thiol groups over intramolecular disulfide bonds to drive the exchange reaction toward intramolecular disulfide bond cleavage. On the other hand, 5 because 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, concentrations in the range of 0.05 to 1.0 M have been found to be effective, and the optimal concentration is considered to be 0.2 M.

The pH of the medium may range from 5.0 to 8.0 when the mRNA is extracted from the cells.

After cell disruption, RNA is separated from cellular proteins and DNA. Several methods have been developed for this purpose.

The usual method 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 followed by centrifugation in Glisin, V., Crvenjakov, R. and Byus, C., Biochemistry 13, 2633 (1974). as described. 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 portion of this RNA is the desired mRNA. Further purification takes advantage of the fact that in cells of higher organisms, 30 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 oligotymidylate has been added, cf. Avis, H. and Leder, P. edel-35 lä. The method described above is suitable when virtually pure, undamaged and translatable mRNA from sources rich in RNase is required. Purification of the mRNA and subsequent precautions can be performed in virtually the same way for any mRNA regardless of the source organism.

In certain cases, such as when using tissue culture cells as a source of mRNA, the RNase impurity may be so low that the RNase inhibition 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 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 using mRNA as a template can be used. The reaction can be performed under previously known conditions using mRNA as a template and a mixture of four deoxynucleoside triphosphates as a DNA strand precursor. It is preferred that one of the deoxynucleoside triphosphates is labeled at the Q 1 position with a P atom, as it allows the reaction to be monitored, serves as a marker in separation methods such as chromatography and electrophoresis, and allows quantitative conclusions to be drawn, cf.

Efstratiadis, A., et al., Supra.

As shown, the reaction result of the reverse transcriptase 30 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.

65447

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

After alkaline hydrolysis and neutralization, the 32 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 polymerase or reverse transcriptase. Reaction conditions are 32 as previously described, including Cfc-P-labeled nucleoside diphosphate. Reverse transcriptase is obtained from several sources. The avian myeloblastosis virus is a preferred source. The virus is manufactured by Dr. D. J. Beard, of Life Sciences Incorporated, St. Petersburg, Florida, under an agreement with the National Institutes of Health.

Once the cDNA 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 hairpin structure can be transformed into 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 the S1 nucleus 30 isolated from Aspergillus oryza (manufactured by Miles Research Products, Elkhart, Indiana). When the DNA hairpin structure is treated with S1 nuclease, molecules with base-pairs are obtained in good yield. Extraction, chromatography and ethanol precipitation are then performed as described above. Efstratiadis et al. 35 have described in the above publication the synthesis of double-stranded cDNA transcripts of mRNA by reverse transcriptase and S1 nuclease.

2θ 65447

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 exonuclease activity of the enzyme and the activity of polymerase-5 acts so that the protruding 3 'end is removed and the protruding 5' end is filled. This ensures that as many cDNAs as possible are involved in the ligation reactions of the next step.

In the next step 10 of the method of the invention, the ends of the cDNA product are processed so as to obtain a restriction endonuclease recognition site sequence 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-15 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 Hind III. Hind III is isolated from Haemophilus influenzae and purified by the following method: Smith, H. 0. 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 from Haemophilus suis, 25 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.

For ligation of the double cDNA, 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, cf.

Heyneker, H. L. et al., And Scheller, R.H. et al., supra. Several such recognition sequence sequences are available to those skilled in the art, and for this reason it is possible to select the ends of the 35 double cDNAs to be suitable for the restriction endonuclease required in each case.

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The insertion of restriction site sequences into the ends of the cDNA can be accomplished by a method called blunt end insertion and is catalyzed by DNA ligase purified by the following method: Panet, A. et al., Biochemistry 12, 5045 (1973). The aforementioned Sgaramel-la, V. et al. have described the blunt-end coupling reaction. Insertion at the blunt end, where the reaction occurs with a blunt-ended cDNA and a large molar excess of a double-stranded decanucleotide containing a Hind III endo-10 nuclease recognition site, yields a product cDNA with a Hind III restriction site sequence at each end.

When the reaction product is treated with Hind end endonuclease, cleavage occurs at the restriction site and single-stranded self-complementing 5 'ends are formed, as shown in Figure 1.

15 4. Construction of a recombinant DNA transfer vector

In principle, a wide variety of viral and plasmid DNA molecules could be used to generate recombinants with cDNA prepared as just described. The main conditions are that the DNA transfer vector is able to enter and replicate in the host cell, and the vector should have a genetic trait that can distinguish the host cells that have received the vector. However, for general safety reasons, the selection should be limited to transfer vector species suitable for the test type in accordance with NIH guidelines 25, cf. above.

Suitable transfer vectors currently approved for use include e.g. several bacteriophage lambda derivatives (see e.g. Blattner, F.R., Williams, B.G., Blechl, A.E.,

Denniston-Thomson, K., Faber. H.E., Furlong, L.A., Grunwald, 30 D.J., Kiefer, D.O., Moore, D.D., Schumm, J.W., Sheldon, E.L.

and Smithies, O., Science 196, 161 (1977)) and col E1 plasmid derivatives (see, e.g., Rodriquez, RL, Bolivar, S., Goodman, HM, Boyer, HW, and Betlach, MN, INC-UCLA Symposium on Molecular Mechanism in the Control of Gene Expression, 35 DP Nierlich, WJ Rutter, CF Fox, Eds. (Academic Press, NY, 1976) pp. 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 of 20 to 40 per host cell, but can be raised to one thousand or more. 5 even greater when host cells are treated with chloramphen-Koli. The ability of a host cell to increase the amount of gene it contains allows, under certain conditions, for the host cell to produce primarily proteins encoded by 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, pBR-317 and pBR-322, which contain the tetracycline resistance gene and the ampicillin 15 resistance gene. The presence of the resistance-causing gene provides a suitable means of selecting cells 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. Within this example, a plasmid derived from col E1 containing e.m. resistance gene and one Hind III recognition site.

23 6 5 4 4 7

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

C

p3R322 has a molecular weight of 2.7 x 10 daltons (Bolivar F., 5 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: Hind III, Sal I, and Bam HI. In addition, pBR322 contains one Eco RI 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 Hae III recognition sites. identification of the face. The recognition sites are labeled on the circular plasmid pBR322 on page 103

R

Bolivar et al. In. The Aβ gene is lost when the plasmid is cleaved from the Pst I recognition site and ligated

R

foreign DNA into it. Similarly, Te is lost upon cleavage of pBR 322 by Hind III, Val I or Bam I endonases and upon incorporation of foreign DNA into their recognition site.

When DNA is ligated into the Pst I recognition site, recombinant molecules are formed that can be detected from strains that are both ampicillin-sensitive (Aβ) and tetracycline-resistant (Te), as well as by ligating DNA with Hind III, Sal I or Bam HI. Recombination molecules that can be detected in strains that are both ampicillin-resistant and tetracycline-sensitive are formed at the recognition site of. 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. Li-10 Saxon characterization can be supplemented by labeling the transfer 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 15 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).

Plasmid pBR313 has a molecular weight of 5.8 x 10 daltons and contains genes that confer ampicillin resistance (Aβ) and tetracycline resistance (Te). The plasmid 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 84 by Bolivar et al. In. When ligating foreign DNA into Hind III,

At the recognition site of Sai I or Bam HI, the tetracycline resistance-30 tense disappears. Thus, recombinant DNA molecules are found in strains that are both ampicillin-resistant and tetracycline-sensitive. The transfer vector can be characterized based on drug resistance properties, the transferred DNA sequence, the restriction enzyme recognition site map 35, and the molecular weights mentioned above.

25 6 5 4 4 7

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 6 5 has a molecular weight of 3.5 x 10 daltons and contains

R

the gene that causes tetracycline resistance (Te). This plasmid contains one recognition site for each of the following endonucleases: 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 ligating foreign DNA

At the Hind III, Sai I, or Bam Hir recognition site, tetracycline resistance disappears. 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, 20 S.H et al. (Proc. Natl-Acad. Sci. USA 70 (1973) 1293) have described the synthesis and preliminary characterization of this plasmid. The characterization has been completed

Cohen, S.N. et al. (Proc. Natl. Acad. Sci. USA 70 (1973) 32U0

Boyer, H.W. et al. (Recombinant Molecules), Beers, R.F. and 25 Basset, E.G. ed. , Raven Press, New York, p. 13 (1977) and Cohen, S.N. et al. (Recombinant Molecules, p. 91).

0

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

R

and contains tetracycline resistance (Te)

D

gene. The Te gene contains one recognition site for each of the following 30 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 recognition site, and four Hinc II recognition sites. Tetracycline resistance is lost by cleavage of pSCIOI with endo-35 nucleases Hind III, Cal I or Bam HI and insertion of foreign DNA into their recognition site. When a recombinant molecule is added to a DNA Hind III, Sai I or Bam HI recognition site, cultures that are tetracycline sensitive are found. A transfer vector containing foreign DNA fused to one of the three sites mentioned above can be characterized by 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) preparing a 10-mile recognition site map, and (c) determining the sequence of the transferred DNA molecule.

There are many possibilities in selecting a suitable host as well as in selecting a plasmid. E. coli strain X-1776, approved by the NIH using P2 workspaces, has been developed for the methods described in this specification (see Curtiss III, R., Ann. Rev. Microbiol. 30, 507 (1976)). . E. coli RR-1 is suitable when P3 workspaces are available.

Recombinant plasmids are constructed by mixing restriction endonuclease-treated plasmid DNA and cDNA treated accordingly. Plasmid DNA is used in a large molar excess over the cDNA so that the cDNA segments form as few combinations as possible with each other. In previous methods, this has resulted in most of the plasmids in the process being those that do not contain a cDNA fragment. As a result, the selection process has become cumbersome and time consuming. In the past, this problem has been solved by attempting to construct DNA vectors with a restriction endonuclease recognition site in the middle of a suitable marker gene such that insertion of the recombinant Nant divides the gene and at the same time loses the function encoded by the gene.

27 6 5 447

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 plasmid ends generated by the endonuclease and prevents self-ligation of the plasmid DNA. Thus, ring formation 10 and at the same time transformation depend 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 1/104.

The present invention is based on the fact that the reaction catalyzed by DNA ligase takes place between the 5 'phosphate end group of the DNA and the 3' hydroxyl end group of the DNA. In the absence of 5'-phosphate, no coupling reaction occurs. When double-stranded DNAs need to be combined, there are three situations, 20 as shown in Table 1: 28 65447

table 1

Case Reactive compounds Ligase product 1 3 '5' 3 '-5' -ΠΗ H 0 PO- -OPO-- -o? O3u + 1 · * Η0- OPO-- ± zh2o 5 '3' 5 '3' II '3 '5' 3 '5' --011. } I2C3? - ° --- O-P-O- + u n -OH + 30H --- OH HO-- l2 ° 5 '· 3' 5 '3' 111 3 '5'.

_ou uo_ no reaction -OH + KO- 5 '3'

In Table 1, double-stranded DNA is shown schematically as solid parallel lines and their corresponding 5 'and 3f end groups are labeled as hydroxyls (OH) or phosphates (OPO ^ H 2). In case I, both reactive molecules have 5'-phosphate at their ends, and thus the two strands covalently join together. 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 case III, there is no 5 1-phosphate at either reactive end and no coupling reaction occurs.

Thus, the 51-phosphate group must be removed from the end whose attachment to the other is to be prevented.

29 65447 Any method of removing the 5 '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 1 to 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.

15 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 the subfragments obtained by restriction nuclease treatment is greatly improved 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. The 5 'terminal phosphates are a structural prerequisite for the action of DNA ligase subfragments. Thus, ends that do not have a 5 'terminal phosphate cannot be covalently joined. DNA subfragments can only be joined to ends that contain a 5 'terminal phosphate formed as a result of the cleavage effect of the restriction endonuclease on the isolated DNA.

30 65447

The procedure described above prevents the most inappropriate joining reaction, namely # that the two fragments join in 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 growth hormone cDNA code was isolated # recombined with the plasmid and transferred to E. coli. When E. coli was highly replicated, it was found to contain a code for whole rat growth hormone and portions of the precursor peptide and a portion of the untranslated 5 'region.

By the method just described, a gene of a higher organism, including a human gene, can be isolated and purified and transferred to a microorganism in which it replicates. The description describes novel recombinant plasmids containing all or part of the isolated gene. The description describes new microorganisms which have not been found in nature to date and whose genetic structure contains the gene of a higher organism.

65447

Example 1 This example describes the isolation and purification of the DNA sequence of the rat growth hormone structural gene, the synthesis of a transfer vector containing the entire rat growth hormone structural gene, and the construction of a microorganism in which the rat growth hormone structural gene is part of a genetic construct.

In the case of non-human genes, U.S. safety regulations 10 do not require the cDNA to be isolated as pure. Thus, it was possible to isolate the entire cDNA of the rat growth hormone structural gene by electrophoretically isolating DNA containing about 800 base pairs, which was known to correspond to the known 15 amino acid sequence of rat growth hormone. As a source of rat growth hormone mRNA, cultured rat pituitary cells, a GH-1 subclone of the cell line and sold by the American Type Culture Collection, were used; Tashjian, A.H., et al., Endochrinology 82, 342 (1968). When such 20 cells are grown under normal conditions, growth hormone mRNA represents only a small percentage, i.e., about 1-3% of the total poly-A containing RNA. However, the level of growth hormone mRNA was able to be increased by the synergistic effect of thyroid hormones and glucocorticoids. RNA was obtained from 5 x 10 cells grown in suspension culture supplemented with 1 mM dexamethasone and 10 nM L-triiodothyronine 4 days before cell recovery. Polyadenylated RNA was isolated from the cytoplasmic membrane fractions of 30 cultured cells, cf. Martial, J.A., Baxter, J.D., Goodman, H.M. and Seeburg, P.H., Proc. Natl. Acad. Sci. USA 74, 1816 (1977) and Bancroft, F. C., Wu, G. and Zubay, G., Proc. Natl. Acad. Sci. USA 70, 3646 (1973). The mRNA was purified and transcribed into double-stranded cDNA essentially as described in Examples 1, 2 and 3 of Application 810687. Gel electrophoresis fractionation showed a weak but still clear band corresponding to about 800 base pairs of DNA.

Treatment of cDNA transcribed from mRNA of whole cultured pituitary cells with Hha I endonuclease yielded two larger DNA fragments expressed by electrophoretic separation, corresponding to approximately 320 nucleotides (fragment A) and 240 nucleotides (fragment B). When the nucleotide sequences of fragments A and B were analyzed as described in Example 5, it was found that these fragments were indeed part of the rat growth hormone code when compared to published rat growth hormone amino acid sequence data and other known growth hormone sequences, cf.

15 Wallis, M. and Davies, R.V.N., Growth Hormone and

Realted Peptides (Eds., Copecile, A. and Muller, E.E.) ss. 1-14 (Elsevier, New York, 1976) and Dayhoff, M.O., Atlas of Protein Sequence and Structure, 5, suppl. 2, ss. 120-121 (National Biomedical Research Foundation, 20 Washington, D.C. 1976). When the 800 bp double-stranded cDNA was isolated electrophoretically as described above and subjected to similar Hhal endonuclease treatment, two fragments corresponding to fragments A and B in length were found among the main products.

Because rat growth hormone cDNA containing about 800 base pairs was not purified for restriction endonuclease treatment, it was necessary to process the DNA to remove odd single-stranded ends.

Removal of such odd ends occurred prior to electrophoretic separation in a solution containing 25 μl of 60 mM Tris-HCl, pH 7.5, 8 mM MgCl 2, 10 mM β-mercaptoethanol, 1 mM ATP and dATP, dTTP, dGTP and dCTP. (200 μM each). When the mixture was incubated for 10 ml to 35 minutes at 10 ° C with 1 unit of E. coli DNA polymerase I, the protruding 31 ends were removed and the protruding 5 'ends were filled exonucleolytically. Boehringer-Mannheim Biochemicals, Indianapolis, Indiana, sells DNA polymerase I.

A Hind III recognition site was ligated to approximately 800 base pairs of rat growth hormone-5 n in cDNA as described in Example 3 of Application 810687. Plasmid pBR-322, which had a gene conferring resistance to α-silicin and a Hind III site in the gene conferring resistance to tetracycline, was treated with Hind III endonuclease and alkaline phosphate-10 as described in Example 4. An 800 bp rat growth hormone cDNA was ligated into the treated plasmid using DNA ligase as described in Example 3 of Application 810687. The ligase reaction mixture was used to transform E. coli X 1776 solusus pension as described in Example 4 of Application 810687. Recombinant colonies were selected on the basis that they grew on medium containing ampicillin but did not grow on medium containing 20 ug / ml tetracycline. Ten colonies were obtained with a plasmid joined by 20 approximately 800 base pairs cleaved by Hind III cleavage.

A preparative amount of 800 base pairs of rat growth hormone DNA was isolated from recombinant clone pRGH-1 and the nucleotide sequence of the isolated DNA was determined as described in Example 5 of Application 2 810687. The nucleotide sequence found contained a region at the 5 'end where no translation corresponding to rat growth hormone had occurred and a sequence for 26 amino acids present in the growth hormone precursor protein prior to secretion. Table 2 also shows the mRNA sequence derived from the gene sequence. The predicted amino acid sequence corresponds to positions 1 and 8, with the exception of the portions of rat growth hormone described by Wallis and Davies (cf. above), which relate to positions 1-43, 65-69, 108-113, 133-143 35 and 150-190.

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35 65447

Example la. (i) Example 1 was repeated except that plasmid pMB-9 prepared by Rodriguez et al. was used instead of plasmid pBR322 DNA. by the method described above (above). The conditions used were otherwise the same as in Example 1, except that the selection was performed as in Example 4 of application 810687. The DNA fragment ligated into the recombinant plasmid was separated to give a DNA fragment of about 800 base pairs, the nucleotide sequence of which is shown in Table 2.

(Ii) Example 1 was repeated except that plasmid pBR322 DNA was replaced with plasmid pSCIOI DNA prepared by the method of Cohen et al. by the method described in (Proc. Natl. Acad. Sci. USA 70 (1973) 1293). All reaction conditions were the same, and selection was performed in the same manner as using plasmid pMB-9. The DNA fragment ligated into the recombinant plasmid was separated to give a DNA fragment of about 800 base pairs, the nucleotide sequence of which is shown in Table 2.

(Iii) Example 1 was repeated except that plasmid pBR322 DNA was replaced with plasmid pBR313 DNA prepared by Bolivar et al. by the method described (Gene 2 (1977) 75). All conditions used, including the final selection, were the same. The DNA fragment ligated into the recombinant plasmid was separated to give a DNA fragment of about 800 base pairs, the nucleotide sequence of which is shown in Table 2.

Example 1. b. Examples 1 and 1a were repeated except that E. coli RR1 or E. coli HB101 was used instead of E. coli X-1776. The conditions used were the same, and the same results were obtained.

f (65447 36

Example 2 This example describes the isolation and purification of the entire human growth hormone structural gene, the synthesis of a recombinant plasmid containing the entire human growth hormone structural gene, and the production of a microorganism in which the entire human growth hormone gene is part of the genetic construct.

Isolation of human growth hormone mRNA was performed essentially as described in Example 1, but human pituitary tumor tissue was used as the biological source. Five benign human pituitary tumors, which had been surgically removed and rapidly cooled in liquid nitrogen and weighed 0.4-1.5 g each, were thawed and ground at 4 ° C in a solution containing 4 M guanidinium thiocyanate 15 and 1 M mercaptoethanol, buffered to pH 5. The homogenate was layered on 1.2 ml of 5.7 M CsCl containing 100 mM EDTA and centrifuged for 18 hours at 15 ° C at 37,000 rpm in a Beckman with an ultra-centrifuge rotor SW 50.1 (Beckman Instrument 20 Company, Fullerton, California). RNA migrated to the bottom of the tube. Further purification was performed using an oligo-dT column and sucrose gradient sedimentation as described in Examples 1-3 of Application 810687. About 10% of the RNA thus isolated encoded growth hormone, judging by the incorporation of 25 reactive amino acid precursors into the anti-growth hormone precipitation material obtained from wheat germ in a cell-free translation system, cf.

Roberts, B.E. and Patterson, B.M., Proc. Natl. Acad. Sci. USA 70, 2330 (1973). Human growth hormone cDNA was prepared essentially as described in Example 1. Human growth hormone cDNA was fractionated by gel electrophoresis, and a fraction moving approximately 800 nucleotides to the corresponding site was selected for cloning. The selected fraction was treated with DNA polymerase I 37 6544 7 as described in Example 1 and then accompanied by a Hind ΙΙΙ recognition site. The cDNA was then recombined by DNA ligase into plasmid pBR-322 treated with alkaline phosphatase. E. coli X-1776 was transformed with recombinant DNA and the strain containing growth hormone DNA was selected as described in Example 1. A strain containing growth hormone DNA was grown in a preparative amount and growth hormone DNA was isolated for nucleotide sequencing. The cloned DNA of growth hormone was found to contain the nucleotide code of the entire amino acid sequence of human growth hormone. The first 23 amino acids of human growth hormone are H „N-Phe-Pro-Thr-Ile-Pro-Leu-Ser- 10 ^ 20 Arg-Leu-Phe-Asp-Asn-Ala-Met-Leu-Arg-Ala-His-Arg Leu-His

Gln-Leu. The remainder of the sequence is shown in Table 3.

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Example 3a. (i) Example 1 was repeated except that plasmid pBR322 DNA was replaced with plasmid pMB-9 DNA prepared by Rodriguez et al. by the method described above (above). All reaction conditions were the same, except that the final selection was performed according to Example 4. The DNA fragment ligated into the recombinant plasmid was separated as described above to give a DNA fragment containing about .800 base pairs, the nucleotide sequence of which is shown in Example 1.

(ii) Example 1 was repeated except that plasmid pBR322 DNA was replaced with plasmid pSCIOI DNA prepared by the method described by Cohen et al (Proc. Natl. Acad. Sci. USA 70 (1973) 1293).

The conditions used were the same and selection was performed in the same manner as for plasmid pMB-9. The DNA fragment ligated into the recombinant plasmid was separated as described above to give a DNA fragment of about 800 base pairs, the nucleotide sequence of which is shown in Example 1.

(iii) Example 1 was repeated except that plasmid pBR322 was replaced with plasmid pBR313 DNA prepared by Bolivar et al. by the method described (Gene 2 (1977) 75). The conditions used, including the final selection method, were the same. The DNA fragment ligated into the recombinant plasmid was separated as described above to give a DNA fragment of about 800 base pairs, the nucleotide sequence of which is shown in Example 1.

30 Example 3b. Examples 2 and 2a were repeated except that E. coli RR1 or E. coli HB101 was used instead of E. coli X-1776. The conditions used were the same and the same results were obtained.

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Claims (1)

4? A method for producing a bacterium containing a nucleotide sequence encoding growth hormone, characterized in that a) the mRNA is isolated from pituitary cells by homogenization in the presence of a ribonuclease inhibitor, for example about 4 mol / l guanidinium thiocyanate and 0.05 to 1.0 mol / l β-mercaptoethanol solution at pH 5.0 to 8.0, so that no ribonuclease degradation of the mRNA occurs, b) a cDNA having a nucleotide sequence encoding growth hormone is prepared from the obtained mRNA in a manner known per se by means of a reverse transcriptase enzyme. C) inserting the restriction endonuclease recognition site sequences into the ends of the resulting double-stranded cDNA in a manner known per se, wherein the restriction endonuclease has the same enzyme as in the following step d), after which the cDNA is cleaved by said restriction. consists of d) opening the bacterial plasmid vector with a restriction endonuclease, for example Hind III, Hsu I or Eco R1, in a manner known per se, for example by incubation at pH 7.6 at 37 ° C for 2 hours in order to an opened plasmid vector having cohesive ends is obtained, e) the 5 'phosphate end groups of the plasmid vector obtained in d) are hydrolysed by treatment with, for example, alkaline phosphatase at pH 8.0 at 65 ° C for 30 minutes 30, whereby the cohesive ends mentioned in d) f) the plasmid vector treated according to e) and the cDNA obtained in c) are ligated in a manner known per se by incubation in the presence of DNA ligase and ATP, for example at pH 7.6 at 14 ° C for one hour, using and g) mixing a bacterium such as E. coli, for example E. coli X-1776, and the reaction product obtained in f) to obtain the nucleotide sequence encoding the growth hormone within 5 hours. bacterium. 42 6 5 4 4 7 For the preparation of bacteria in which a nucleus sequence is used, in which case a nucleotide sequence, 5 a) mRNA is isolated from the pituitary genome to be homogenized in the presence of a ribonuclear gene t.ex. av en lös-Ning innehällande 4 mol / l guanidinium thiocyanate and 0.05 ... 1.0 10 mol / l / ft-mercaptoethanol, at pH av 5.0 ... 8.0, colors with faces ribonucleassonfallfall av mRNA e), b) ur mRNA framställes pä i och för sig känt sätt med mit tillhjälp av ett reverstransskripttasenzym en sädan cDNA, vilken har en för tillväxthormon kodande nucleo- sequence, och den nämnda cDNAsn omvandlas account The cDNA of the cDNA is limited to the presence of the enzyme as described above. d) the bacterial plasmid vector is present with the restriction endonuclease, t.ex. med Hind III, Hsu I, eller Eco Rl, pä i och för sig känt sätt t.ex. The genome is incubated at a pH of 7.6 and at a temperature of 37 ° C for 2 hours, for which a plasmid vector is used in the cohesive region, e) at point d) a plasmid vector 5'-phosphatide group hydrolyzes the genome .ex. the phosphate phosphate at a pH of 8.0 and a temperature of 65 ° C for 30 minutes, color d) and (d) a cohesive plasmid vector and a mixture thereof; punkt c) erhällna cDNA nplplas account varandra 35 pä i och för sig känt sätt, genom att incubera i närvaro av