FI64953C - FOERFARANDE FOER FRAMSTAELLNING AV EN REKOMBINANT-DNA-MOLEKYL SOM INNEHAOLLER EN FOER TILLVAEXTHORMON KODANDE NUCLEOTIDS SEQUENCE OCH SOM KAN ANVAENDAS VID FRAMSTAELLNING AV EN TILLVAEXTHORMON PRODUCERANDE MIKRO - Google Patents

Description

64953

Method for producing a recombinant DNA molecule containing a nucleotide sequence encoding a growth hormone and used in the production of a growth hormone-producing microorganism 5

This invention relates to a method for producing a recombinant DNA molecule containing a nucleotide sequence encoding a growth hormone and used in the manufacture of a growth hormone-producing microorganism.

The method according to the invention is characterized in that! a) The mRNA is isolated from pituitary cells by homogenization in the presence of a ribonuclease inhibitor, for example a mixture of 4-guanidinium thiocyanate and 0.05 to 1.0-mb mercaptoethanol at pH 5.0. ... 8.0, in which case 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, and said cDNA is double-stranded, c) restriction endonuclease recognition target sequences at 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 to form cohesive ends. , for example by means of 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 to obtain an opened plasmid vector with cohesive ends, _-V "2 64953 e) hydrolyzing the 5'-phosphate end groups of the plasmid vector obtained in d) by treatment for example with alkaline phosphatase at pH 8.0 at 65 ° C for 30 minutes, in which case the cohesive ends mentioned in d) cannot be joined together, f) the plasmid vector treated according to e) and the cDNA obtained in c) are ligated as such by incubating in a known manner 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 10 plasmid vectors to obtain a recombinant DNA molecule containing a nucleotide sequence encoding growth hormone.

The following symbols and abbreviations are used in this description: «f

15 DNA - deoxyribonucleic acid RNA - ribonucleic acid cDNA - complementary DNA

(synthesized enzymatically from the mRNA sequence) mRNA - messenger RNA 20 tRNA - transfer RNA

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

Tris - 2-amino-2-hydroxyethyl-1,3-propanediol 30 EDTA - ethylenediaminetetraacetic acid ATP - adenosine triphosphate TTP - thymidine triphosphate 3 64953 ί

Hind ΙΙΙ recognition site - SEQ ID • A 'AGCTT, specifically a broken arrow into the Hind ΙΙΙ endonucleases 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 - Colchicine E1-forming plasmid poly dA - polydeoxyadenylate 10 oligo flT, 0 ΊΟ - oligodeoxythymidylate (12-18

Iz “lo base)

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 ^ 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 AcCTGG, specifically a broken arrow into the Eco RLI endonucleases.

Search II recognition site - SEQ ID AGCGcirT, AGCGC 'tc 25 GGCGC \ ^ GGCGcJ T or 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.

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

Sal I recognition site - SEQ G ^ TCGAC, specific cleavage of the arrow into the Sal I endonucleases.

4 64953

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 64953 on the 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 V is CZ = A or G if VJ 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 termed the first step in the biological process by which nucleotide sequence-25 information is converted to an amino acid sequence. At this point, the RNA is first copied to 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 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.

Many proteins of medical or research importance are located in or produced by cells of higher organisms, such as vertebrates. These include e.g. peptide hormones such as growth hormone.

15 These proteins are often difficult to isolate in usable amounts, and this problem is quite difficult 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 organisms.

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 cultivate in chemically defined media. Fermentation technology is advanced. It is possible to cultivate organisms quickly and with high yields. In addition, some microorganisms are thoroughly studied and known for their genes and characteristics.

7 64953 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 defining the production of a particular protein to 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 engineered to produce 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 will be described in the light of the 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 ditriphosphates, dATP, dGTP, dCTP, and dTTP. The reaction is initiated by non-covalent binding of the DNA template to the 3 'end of the mRNA and is followed by the gradual insertion of the required deoxynucleotides to the 3' end of the growing chain as base pairs defined by the mRNA nucleotide sequence. The resulting molecule can be considered as a hairpin structure containing the original RNA 64953 linked by a single DNA strand to a complementary DNA strand. Reverse transcriptase is also capable of catalyzing a similar reaction using a single-stranded DNA template, the product obtained 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 that are able to hydrolyze 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 several different sources and characterized by the nucleotide sequence of their recognition site.

Some restriction endonucleases hydrolyze phosphodiester bonds at 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. Roberts, R.J. Crit. Rev. Biochem. 4, 123 (1976). However, hestriction sites are quite rare, but the utility of restriction 9 64953 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, 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 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-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, 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 ___ - · r ~ 10 64953 in a microbial cell and does not belong to the genome of the host cell ram. Plasrai is not genetically bound to the chromosome of the host cell. Plasmid DNA exists as annular double-stranded molecules, usually with a molecular weight of a few million 5, some even more than 10, and usually represent only a small percentage of the total DNA of the cell. Plasmid DNA can usually be separated from host DNA due to its large size. Plasmids can replicate regardless of the rate of division of the host cell, and sometimes their rate of division 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 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 suitable for selection purposes, such as genes that confer drug resistance.

The ability to transfer the genetic code of a protein essential for the normal metabolism of a particular higher organism to a microorganism, such as a bacterium, opens up significant possibilities for the production of such proteins by culturing. This, in turn, offers significant potential for increasing or replacing such proteins with proteins produced by transformed microorganisms when the ability of a higher organism to produce such proteins is deficient, and one might consider, for example, the possibility of establishing a symbiotic relationship with the microorganism synthesized by this invention. between the organism and a human suffering from a chronic or acute deficiency disease, wherein the microorganism genetically transformed as described herein would be inoculated or otherwise linked to a human to compensate for a pathological deficiency in human metabolism.

As examples of the specific DNA sequences of this invention, the structural gene for rat growth hormone and human growth hormone is transferred into a bacterium.

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 reverse transcriptase-derived product is subjected to a procedure for selectively deleting the ribonucleotide sequence. The remaining deoxynucleotide sequence complementary to the original mRNA is incubated in a second reaction with reverse transcriptase or 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 strand 12 64953, which cleaves the simple linking strand. The resulting double-stranded cDNA is extended by adding to each end a specific DNA containing the restriction enzyme recognition site sequence.

5 The addition is catalyzed by the enzyme DNA ligase. 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 with 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: 1. 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 in certain places 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 the cell and method of isolation to be isolated can take into account the advantages offered by the initial degree of purity of the mRNA to be isolated.

In most tissues, glands, and organs, cell-5 and connects fibrous connective tissue, which 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. The methods of this invention can be applied to isolate different types of cells from a variety of tissues.

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 culturing at a particular temperature and / or a particular nutrient or other chemical agent. In 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 whole molecule incapable of transferring the intact genetic sequence to the micro-

II

15 64953 organisms. 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 contaminates organic chemicals.

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 RNA.

10 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, diiodo-15 salicylate, etc. The relative effectiveness of the salts formed by such anions and cations is determined by their solubility. For example, lithium diiodic 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 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 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.

The efficacy of a particular chaotropic salt is directly related to its concentration when it is desired to inhibit RNase in the extraction of RNA from cells or tissues. Affordable concentration

______ TTT

16 64953 is therefore the highest practicable concentration. 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 10 often depends on the ratio of the denaturant concentration to the threshold value of 5-10 raised to the power, cf. Tan-ford, C. A., Adv. Prot. Chem. 23, 121 (1968). Qualitatively, this ratio means that only a slightly more efficient denaturant than guanidinium hydrochloride can denature protein-15 many times faster at the same concentration. The relationship between the kinetics of RNase naturalization 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 that has a low threshold concentration in denaturation and high water solubility. Therefore, guanidinium thiocyanate is more preferable than lithium diiodal 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-cleavage 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 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 having 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 to break 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, concentrations in the range of 0.05 to 1.0 M 10 have been found to be effective, and the optimal concentration is considered to be 0.2 M.

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.

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 and then centrifuge as described in Glisin, V., Crvenjakov 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 the mRNA of higher organisms is processed after transcription by the addition of polyadenylic 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 64953 18 Γ is required. Purification of the mRNA and subsequent in vitro procedures can be performed in virtually the same manner on 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 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 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 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 <Z-32 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., Supra.

As can be seen, 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 the cDNA has been synthesized enzymatically, the RNA template can be removed. Several methods are known for which selective degradation of RNA-35a RNA in the presence of DNA.

Early hydrolysis is a preferred method because it is very selective and can be very easily adjusted by pH.

32

After alkaline hydrolysis and neutralization, the P-labeled 19 64953 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 as previously described, including Pt-labeled nucleoside triphosphate. Reverse transcriptase is obtained from several sources. The avian myeloblastosis virus is a preferred source. The virus is manufactured by Dr. D. J. Beard, Life Sciences Incorporated,

St. Petersburg, Florida, National Institutes of Health folder sa by agreement.

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 it is desired to obtain DNA 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 the intrinsic portions of DNA yk-20. A well-suited enzyme for this purpose is S1 nuclease isolated from Aspergillus oryzae (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. have described in the above publication the synthesis of double-stranded cDNA transcripts of mRNA by reverse transcriptase and S1 nuclease.

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 polymerase activity works so that the protruding 3 'end is removed and the protruding 5' end is filled. This ensures that the largest possible number of cDNAs are involved in the next step ligation reactions.

In the next step of the method of the invention, the ends of the cDNA product are processed so as to obtain a restriction thioendonuclease recognition site sequence at each end. Practical reasons determine the choice of DNA fragment to be inserted at the ends. The sequence to be inserted at the ends is selected on the basis of the restriction endonuclease, which in turn is selected on the basis of 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 Hind 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 from Haemophilus suis, Hsu I, catalyzes the same site-15 specific hydrolysis at the same recognition site as Hind III. Thus, these two enzymes can be considered as functionally alternative.

For ligation of the double cDNA, it is preferred to use a chemically synthesized double-stranded decanucleo-20-tide containing a Hind III recognition site. The sequence of the double-stranded decanucleotide is shown in Figure 1, cf. Hey-neker, H. L., et al., And Scheller, R. H. et al., Supra. 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-25 DNA to be suitable for 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 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 to 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 product of reaction 21 64953 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.

4. Construction of a Recombinant DNA Transfer Vector 5 Suitable transfer vectors currently acceptable for use include e.g. several bacteriophage lambda derivatives (see e.g. Blattner, FR, Williams, BG, Blechl, AE, Denniston-Thompson, K., Faber, HE, Furlong, LA, Grun-Wald, DJ, Kiefer, D. 0., Moore, DD , Schumm, JW, Sheldon, EL, and Smithies, 0., Science 196, 161 (1977)) and Col El plasmid derivatives (see, e.g., Rodriquez, RL, Bolivar, S., Goodman, HM, Boyer, HW, and Betlach, M.

N., INC-UCLA Symposium on Molecular Mechanism in the Control of Gene Expression, D. P. Nierlich, W. J. Rutter, C. F. Fox, 15 Eds. (Academic Press, NY, 1976) ss. 471 - 477). Plasmids derived from Col E1 are characterized by a relatively small size, having a molecular weight of the order of a few million, and the fact that under normal conditions the number of copies of plasmid DNA per host cell is 20-40, but can be raised to a thousand or more. when the host cells are treated with chloramphenicol. The ability of a host cell to increase the amount of gene it contains, under certain conditions, under the control of a researcher, allows the host cell to produce primarily proteins encoded by plasmid genes.

Accordingly, 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 resistance gene. The presence of the resistance ______ - τ ~ _ 22 6 4 9 5 3 gene provides a suitable means to select 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. In the examples contained in this specification, a plasmid derived from col Elr containing e.m. resistance gene and one Hind III recognition site.

Plasmid pBR-322 has been highly characterized.

10 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

sin (Aβ) and tetracycline resistance (Te) 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, 20 five Eco RII recognition sites, three Bgl I recognition sites, 12 Alu I recognition sites, 12 Search II recognition sites and 17 Search III: n identification point. The recognition sites are labeled on the circular plasmid pBR322 on page 103

Bolivar et al. In. The Aβ gene is lost upon cleavage of plasmid 25 from the Pst I recognition site and ligation

R

foreign DNA into 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.

When DNA is ligated into the Pst I recognition site, recombinant molecules are formed that can be detected in strains that

S

are both ampicillin-sensitive (Aβ) and tetracycline-ni-resistant (Te), and by inserting DNA into the Sai I or Bam HI recognition site of Hind III, recombination molecules are formed that can be detected in strains that are both ampicillin-resistant and tetracycline-positive. 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-resistant. 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. Therefore, the characterization of Li-10 can be supplemented by labeling different endonuclease recognition sites 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).

g 20 Plasmid pBR313 has a molecular weight of 5.8 x 10 daltons,

R

and contains genes causing 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.

24 64953

The third plasmid that has been highly characterized is pMB 9. This plasmid was prepared according to Rodriguez, R.L. et al. (supra) and Bolivar, F., et al., (Gene 2 (1977) 75). Plasmid pMB 9 g 5 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 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

Hind III, got I or Bam HI 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.

Several vectors can be used to transform 20 Bacillus subtilis. These vectors are derived from the microorganism Staphylococcus aureus (see Fordanescu, S., J. Bakteriol. 124 (1974) 597 and Ehrlich, S.D., Proc. Natl. Acad. Sci. USA, 74 (1977) 1680). These plasmids have specific resistance and specific restriction endonuclease sites. For example, plasmid pC194 confers resistance to chloramphenicol (Cm), contains a single Hind β site, and has a molecular weight g of 1.8 x 10 daltons. The foreign DNA thus binds to the HindIII site of plasmid pC194. The transfer vector containing the 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, compared to the molecular weight of the starting materials. The sequence of the ligated DNA can also be determined.

35 There are many possibilities for selecting a suitable host as well as for selecting a plasmid.

Il 25 6 4 9 5 3 E. coli strain X-1776, approved by the NIH when using P2 workspaces, has been developed for the methods described in this specification, cf. Curtiss III, R., Ann, Rev. Microbiol. 30, 507 (1976). E. coli RR-1 is suitable when P3-5 working spaces are available.

Recombinant plasmids are constructed by mixing restriction endonuclease-treated plasmid DNA with cDNA terminated accordingly. Plasmid DNA is used in large molar excess over cDNA to minimize combinations of cDNA segments with each other. In previous methods, this has resulted in most of the plasmids in circulation 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 the insertion of a recombinant Nant splits the gene and at the same time loses the function encoded by the gene.

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 'ends from the ends of the plasmid generated by endonuclease and prevents self-ligation of the plasmid DNA. Thus, ring formation and concomitant trans-30 formation depends on the incorporation of a DNA fragment containing 5'-phosphorylated ends. The method described reduces the relative incidence of non-recombinant transformation to less than 1 to 10.

This invention is based on the fact that the reaction catalyzed by DNA ligase takes place between the 5'-phosphate end group of DNA and the 3'-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: ____ .. τ: - 26 64953

table 1

Case Reactive compounds Ligase product 5 I 3 '5' 3 '5' -OH II 0 PO- --OPO-- -OPO, IL + 2 3H0- -OPO - i-2H2o 5 '3' 5 '3' II 3 ' 5 '3' 5 '- Was H-0,? - 0 --- OPO- 10 -OH + 3OH --- OH HO - ^ * U2 ° 5' · 3 '3' 3 'III 3' 5 ' _OH HO_ no rc ^ thio -OH + HO- 15 5 '3 ·

In Table 1, the 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 (OPOgH 2). thus, the two strands are covalently joined together. In case II, only 25 of the ends to be joined contain 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 'phosphate at either reactive end and no coupling reaction takes place.

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

64953 27 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 it into 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 subfragments obtained by restriction-20 endonuclease 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. 5′-terminal phosphates are a structural prerequisite for the DNA ligase subfragment binding activity. 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.

28 64953

The procedure described above prevents the most inappropriate joining reaction, namely that the two fragments would 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 take place 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 had replicated. vigorously, a sequence of about 800 nucleotides was isolated and found to contain a code for whole rat growth hormone as well as portions of the procursor peptide and a portion of the untranslated 5 'region.

By the method just described, it is possible to isolate a gene of a higher organism, including a human gene, and transfer it to the microorganism in which it is present. The description describes novel recombinant plasmids containing all or part of the isolated gene. The report describes new micro-organisms that have not been found in nature to date and whose genetic structure contains the gene of a higher organism. The following examples describe recombinant plasmids containing portions of the rat growth hormone gene and the human growth hormone gene, as well as 30 novel microorganisms containing said genes.

Il 29 64953

Example 1 This example describes the isolation and purification of the DNA sequence of the rat growth hormone structural gene.

In the case of non-human genes, safety regulations do not require the isolation of cDNA in a particularly pure manner. Thus, it is possible to isolate the entire cDNA of the rat growth hormone structural gene by electrophoretically isolating DNA containing about 800 base pairs, which is known to correspond to the known amino acid sequence of rat growth hormone. As a source of rat growth hormone mRNA, cultured rat pituitary cells, which were a subclone of the cell generation GH-1 and sold by the American Type Culture Collection, were used; Tashjian, A.H., 15 et al., Endochrinology 82, 342 (1968). When such 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 a 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 25 RNA was isolated from the cytoplasmic membrane fraction of cultured cells, cf. Martial, J.A., Baxter, J.D., Goodman, H.M. and Seeburg, P.H., Proc. Natl. Acad. Sci.

U.S. 74, 1816 (1977) and Bancroft, F. C., Wu, G. and Zubay, G., Proc. Natl. Acad. Sci. USA 70, 3646 (1973).

30 mRNA was purified and transcribed into double-stranded cDNA (see Examples 1-3 of the parent application). Gee large electrophoresis fractionation showed a weak but clear band corresponding to DNA of about 800 base pairs in length.

30 64953

Treatment of cDNA transcribed from the whole mRNA of 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 of the parent application, it was found that these fragments were indeed parts of the rat growth hormone code when compared to published 10 rat growth hormone amino acid sequence data and other known growth hormone sequences, cf. 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., 15 Atlas of Protein Sequence and Structure, 5, suppl. 2, ss. 120-121 (National Biomedical Research Foundation, Washington, D.C. 1976). When the 800 bp double-stranded cDNA was isolated electrophoretically as described above and subjected to the same Hha I 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 25 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 30 μm mercaptoethanol, 1 mM ATP, and dATP, dTTP, dGTP

and dOTP (200 μM each). After incubation for 10 minutes at 10 ° C with 1 unit of E. coli DNA polymerase I, the protruding 3 * ends were removed and

B

64953 31 protruding 5 'ends filled exonucleolytically. Boehringer-Mannheim Biochemicals, Indianapolis / Indiana, sells DNA polymerase I.

A rat growth hormone cDNA containing approximately 800 base pairs was ligated with a HindIII recognition site as described in Example 5 of the parent application. Plasmid pBR-322, which had an ampicillin resistance gene and a HindIII site in the tetracycline resistance gene, was treated with Hind III endonuclease and alkaline phosphatase as described in Example 4 of the parent application. The 800 plasmids were ligated to the treated 10 plasmid by rat growth hormone cDNA as described in Example 3 of the parent application. The ligase reaction mixture was used to transform E. coli X-1776 cell suspension as described in Example 4 of the parent application. Recombinant colonies were selected on the basis that they grew on medium containing ampicillin but did not grow on medium containing 20 μg / ml tetracycline. Ten colonies were obtained with a plasmid joined by an approximately 800 bp fragment cleaved by Hind III cleavage.

A preparative amount of 20,800 bp 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 the strain application. The nucleotide sequence found contained at the 5 'end a region where no translation corresponding to rat growth hormone had occurred and a sequence for 26 amino acids present in the growth hormone pre-cursor protein prior to secretion. Table 1 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 the 30 well rat growth hormone described by Wallis and Davies (cf. above), which relate to positions 1 to 43, 65 to 69, 108 to 113, 133 to 143 and 150 to 190.

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33 64953

Example 2

Example 1 was repeated except that the DNA of plasmid pBR-322 was replaced by DNA of plasmid pMB-9 (method of preparation: Rodriquez, RL, Bolivar, F., Good-5 man, HM, Boyer, HW and Betlach, M., INC- The UCLA Symposium is Molecular and Cellular Biology, DP Wierlich, WJ Rutter, and CF Fox, Eds. (Academic Press, New York 1976), pp. 471-477). The conditions used were otherwise the same as in Example 1, except that the selection was performed as in Example 4 of the 10 strain applications. 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 1.

Example 3 This example describes the isolation and purification of the entire structural gene for human growth hormone.

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, surgically removed and rapidly cooled in liquid nitrogen, weighing 0.4 to 1.5 g each, were thawed and ground at 4 ° C in a solution containing 4 M guanidinium thiocyanate and 1 M mercaptoethanol. , and which was buffered to pH 5.

34 6 4 9 5 3

The homogenate was deposited in 1.2 lanes with 5.7 M CsCl containing 100 mM EDTA and centrifuged for 18 hours at 15 ° C at 37,000 rpm on a Beckman ultra-centrifuge rotor SW 50.1 (Beckman Instrument 5 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, 2 and 3. Approximately 10% of the RNA thus isolated encoded growth hormone, judging by the incorporation of 10 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 amplified 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 as described in Example 1 and then ligated with Hind III recognition sites. The cDNA was then recoxified by DNA ligase to plasmid pBR-322 treated with alkaline phosphatase. E. coli X-1776 was transformed with recombinant DNA and a strain containing 25 growth hormone DNAs was selected. A strain containing growth hormone DNA was grown in a preparative amount and growth hormone DNA was isolated for nucleotide sequencing. The cloned growth hormone DNA was found to contain the entire amino acid 30 nucleotide sequence of human growth hormone. The first 23 amino acids of human growth hormone are H2N-Phe-Pro-Thr-Ile-Pro-Leu-Ser-Arg-Leu-P? I§-Asp-Asn-Ala-Met-Leu-Arg-Ala-His-Arg- Leu-His-Gln-Leu. The remainder of the sequence is shown in Table 2.

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Example 4.

Example 3 was repeated except that plasmid pBR-322 DNA was replaced with plasmid pMB-9 DNA prepared by Rodricjuez et al. by describing the go-5 method (above). All reaction conditions were the same, except that the final selection was performed according to Example 4 of the parent application. The DNA fragment ligated into the recombinant plasmid was separated as described above to give a DNA fragment containing about 800 base-10 pairs, the nucleotide sequence of which is shown in Example 3.

Claims (1)

A method for producing a recombinant DNA molecule containing a nucleotide sequence encoding a growth hormone and used in the manufacture of a growth hormone-producing microorganism, characterized in that a) mRNA is isolated from pituitary cells 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 / 5-mercaptoethanol at pH 5.0 to 8.0, so that no ribonuclease degradation of the mRNA occurs, b ) a cDNA having a growth hormone-encoding nucleotide sequence is prepared from the obtained mRNA in a manner known per se by means of a reverse hormone-encoding nucleotide sequence, and said cDNA is double-stranded, c) 20 is the same enzyme as in d) below, after which the cDNA is cleaved by said restriction endonuclease to form cohesive ends, d) opening the bacterial plasmid vector with a restriction endonuclease, for example Hind III, Hsu I 25 or Eco R1, in a manner known per se, e.g. by incubation at pH 7.6 37 E) to hydrolyze 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, d) said cohesive ends cannot be joined together, 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 »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 growth hormone. 39 64953 For the production of a recombinant DNA molecule comprising a nucleus-5 nucleotide sequence and for the production of a production of a microorganism produced by a recombinant DNA hormone, the gene for which the isolate is isolated dessa i närvaro av ett ribonucleas inhiberande 10 ämne, vilket kan utgöras t.ex. in blanding with 4-m-guanidinium thiocyanate and 0.05 ... 1.0-m / i-mercaptoethanol, at pH av 5.0 ... 8.0, colors on the face ribonuclease-fall av mRNA ej sker, b) ur den erhälinä mRNA framställes pä i och för 15 sig känt sätt med tillhjälp av ett reverstransskriptasenzym en hundred hundred cDNA, vilken har en för tillväxthormon codand nucleotide sequence, och nämnda cDNA's omvandlas account tvä-trä The cDNA of the transgene cDNA has a restriction enzyme as described above, and the cDNA is cleaved with this step. the bacterial plasmid vector is shown with the restriction endonuclease, t.ex. with Hind III, Hsu I el-ler Eco Rl, pä och i för sig känt sätt t.ex. the genome is inoculated at a pH of 7.6 and at a temperature of 37 ° C and 2 times the amount of the plasmid vector immediately present, (e) the point d) of the plasmid vector 5'-phosphate group hydrolyser genome behandling med t.ex. the alkaline phosphate is at a pH of 8.0 and at a temperature of 65 ° C for 30 minutes, the color of point d) is readily adjusted to 35 ° C in the case of a variant, (f) is a point of the plasmid vector and punkt c) erhällna cDNA kopplas account varandra pä