FI74732B - DNA-OEVERFOERINGSVEKTOR, VILKEN INNEHAOLLER EN NUCLEOTIDSEKVENS SOM KODAR FOER TILLVAEXTHORMON. - Google Patents

Description

1 74732

A DNA transfer vector containing a nucleotide sequence encoding growth hormone

This invention relates to a DNA transfer vector containing a nucleotide sequence encoding a growth hormone which can be used to transform a microorganism.

according to the invention, the DNA transfer vector is a 10 characterized in that it contains the human growth hormone encoding a deoxynucleotide sequence which is as fol-ruled: 5 'ttk1ccl2acl3atm4ccl5x6ty6qr7s7w8gz8x9ty9ttk10gak1, aak12 GCL13ATG14X15TY15W16GZ16GCL17CAK18W19GZ19X20Ty20CAK21CAJ22X23TY23 15 GCL24TTK25GAK26ACL27TAK28CAJ29GAJ30TTK31GAJ32GAJ33GCL34TAK35ATM36 CCL37AAJ38GAJ39CAJ40AAJ41TAK42QR43S43TTK44X45TY45CAJ46AAK47CCL48 CAJ49ACL50QR51s51x52TY52TGK53TTK54QR55S55GAJ56QR57S57A ™ 58 ccl59acl60ccl61qr62s62aak63w64gz64gaj65gaj66acl67caj68caj69aaj70 QR71S71AAK72X73TY73GAJ74X75TY75X76TY76W77GZ77A ™ 78QR79S79X80TY80X81 2C TY81X82TY82A ™ 83CAJ84QR85S85TGG86X87TY87GAJ88CCL89GTL90CAJ91 TTK92X93TY93W94GZ94QR95S95GTL96TTK97GCL98AAK99iR100S100X101TY101 GTL1 02TAK103 ^ 104GCL105QR106S106GAK107QR108S108 ^ 109GTL110 TAK111GAK112X11 3TY11 3X11 4TY11 4 * ^ 11 5GAK11 6X11 7 ™ 11 7GAJ118GAJ11 9 GGL120A ™ 121CAJ122ACL123X124TY124ATG125GGL126W127GZ127X128TY123T 8TTK1 39AAJl 40CAJ1 41ACL1 42TAK1 43QR1 44S1 44AAJ1 45TTK1 46 GAK1 47ACL1 * 48 ^ 1 ^ 49QR150S150CAK15V52 153GAK154GCL155 X156TY156X157TY157AAJi58 *** 159TAK160GGL161X162TY162X163TY163TAK164 TGK16 5TTK166W16 7GZ16 ^^^ 168GAK16 9ATG170GAK171 7 1 7 7 2GTL1 3GAJ1 April 7, 30 ACL175TTK176X4 7yTY177W4 7gGZ178aTM17gGTL18QCAJ181TGK102W183 GZ183QR184S184GTL185GAJ186GGL187QR188S188TGK189GGL190 ttk191 --- 3 'wherein 2 74732 A is deoxyadenyl, G is deoxyganylyl, C is deoxycytosyl, T is thymidyl, J is A or G, K is T or C, L is A, T, C or G, M is A, C or T, X is T or C if Y is A or G, and C if Y n J nn 10 is C or T, Y is A, G, C or T if X is C, and A or G, n J n if X is T, n

Wn is C or A if Z is G or A, and C if Z is C or T, n Z is A, G, C or T if W is C, and A or G, nn if W is A, J n QRn is TC if Sn is A, G, C or T, and AG if S is T or C, n

Sn is A, G, C, or T if QRn is TC, and T or C, 20 if QR is AG, n, and extension n denotes the position of the amino acid of human growth hormone corresponding to the nucleotide sequence according to the genetic code and the amino acids are numbered from the amino terminus.

25 The following symbols and and abbreviations are used in this explanation:

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

30 (enzymatically synthesized from the mRNA sequence)

mRNA - messenger RNA tRNA - transfer RNA

dATP - deoxyadenosine triphosphate dTTP - deoxythymidine triphosphate 35 dGTP - deoxyguanosine triphosphate dCTP - deoxycytidine triphosphate A - adonin, T - thyrine, G - guanine 3 74732 C - cytosine

Tris - 2-amino-2-hydroxyethyl-1,3-propanediol EDTA - ethylenediaminetetraacetic acid ATP - adenosine triphosphate 5 TTP - thymidine triphosphate

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

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

Col El - colicin E1-forming plasmid poly d A - polydeoxyadenylate 15 oligo d Ti2-18 “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 G | GATCC, specifically the direction of arrow 20 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 25 into the Eco RI endonuclease means.

Eco RII recognition site - sequence icCAGG or IcCTGG, specifically. a broken arrow into the Eco RII-endonukle-ass form.

Search II recognition site - SEQ ID AGCGcIt, AGCGC 'tc 30 GGCGC \ It GGCGC A 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.

35 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 74732 4

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 poorly understood. 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

25 Penylalanine (Phe) RTD Histidine (His) CAK

Leucine (Leu) XTY Glutamine (Gin) CAJ

Isoleucine (Ile) ATM Asparagine (Asn) AAK

Methionine (Met) ATG Lysine (Lys) AAJ

Välin (Vai) GTL Asparagine

30 Serine (Ser) QRS acid (Asp) GAK

Proline (Pro) CCL Glutamine

Threonine (Thr) ACL acid (Glu) GAJ

Alanine (Lower) GCL Cysteine (Cys) TGK

Tyrosine (Tyr) TAK Tryptophan (Try) TGG

35 Terminal signal TAJ Arginine (Arg) WGZ

Terminal signal TGA Glycine (Gly) GGL

5,74732

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

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

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

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

Y: A or G if X is T

W = C or A if Z is A or G

W = C if Z is C or T

Z = A, G, C or T if W is C, and

Z = A or G if W is A QR = TC if S is A, G, C or T QR = AG if S is T or CS = A, G, C or T if QR is TC 20 S = T or C if QR is AG

J = A or G

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 converted to an amino acid sequence. In this step, the RNA is first copied with the DNA segment whose sequence defines the protein to be produced. RNA is a polynucleotide similar to DNA except that deoxyribose has been replaced with ribose and uracil is used in place 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 the cell, mRNA is used as a template in the complex process involving numerous ent-5 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 many. 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 keep the cell line constant so that the desired specific properties are not retained.

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 are thoroughly studied and known for their genes and characteristics.

Thus, it is highly desirable to be able to transfer the genetic code of a medically significant protein from an organism that normally renders the protein to a suitable microorganism. In this way, the microorganism can synthesize the protein under controlled growth conditions 7,74732 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 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 to which the invention relates is a multi-step process involving enzyme-catalyzed reactions. The nature of these enzyme reactions will be explained on the basis of facts known to date.

Reverse transcriptase (DNA polymerase) catalyzes the synthesis of DNA complementary to the RNA template strand in the presence of an RNA template, a DNA template, and four deoxy-20 nucleoside triphosphates, 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 into 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 linked by a single strand of DNA to a complementary strand of DNA. Reverse transcriptase is also capable of catalyzing a similar reaction using a single-stranded DNA template, the product obtained being a double-stranded DNA hairpin with strands at one end joined by single-stranded DNA, cf. Aviv, H. and Leder, P., Proc. Natl.

Acad. Sci. USA 69, 1408 (1972) and Efstratiadis, A., Kafatos, F.C., Maxam, A.F. and Maniatis, T., Cell 7, 279 (1976). Restriction endonucleases are enzymes capable of hydrolyzing phosphodiester bonds in double-stranded DNA to form a cleavage site in the DNA strand. If the DNA is in the form of a closed loop, the structure of the loop becomes linear. The main property of this type of enzyme is that its hydrolytic effect is limited to a site with a specific nucleotide sequence. This sequence is called the restriction endonuclease recognition site. Restriction endonucleases have been isolated from a variety of sources and characterized by the nucleotide sequences of their recognition sites. Some restriction endonucleases hydrolyze phosphodiester-10 crosslinks from the same site in each strand to form a blunt end. Some catalyze the hydrolysis of bonds separated by a few nucleotides, creating a free un-internalized region at each end of the molecule. Such single-stranded ends are self-complementary and thus cohesive and can be used to anneal 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, 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 necessary restriction oligonucleotide to the ends of the molecule, exposing the product to the required restriction endonuclease to create the necessary cohesive ends, cf. 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, 35 RH, Dickerson, RE, Boyer, HW, Riggs, AD and Itakura, K., Science 196, 177 (1977).

9 74732 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).

5 DNA ligase is an enzyme capable of catalysing the formation of a phosphodiester bond in the earth between two segments of DNA containing 5'-phosphate and 3'-hydroxyl, respectively, and may be composed of two DNA fragments held by cohesive ends. 10 together. The normal mode of action of an enzyme is considered to be that it combines several daughter DNA fragments generated in parallel with another strand. However, DNA ligase can, under appropriate conditions, catalyze the fusion of blunt ends in which two blunt-ended Mole-15 rabbits 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 said multi-step method, a particular DNA fragment is inserted into a DNA vector, such as a plasmid. Plasmid is the name used for any independently replicating unit of DNA that is present in a micro-bis 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

Q

million, some even over 10, and usually represent only a small percentage of the total DNA of the cell. Plasmid DNA can usually be separated from host cell DNA due to its large size difference. Plasmids can replicate regardless of the rate of proliferation of the host cell, and sometimes their 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 replication capacity and gene expression capacity are not substantially altered. Thus, the plasmid serves as a useful vector to transfer a DNA segment to a new host cell. Plasmids suitable for Re-5 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 metabolism of a particular higher organism to a microorganism, such as a bacterium, opens up significant possibilities for the production of such proteins by culture. This, in turn, offers significant opportunities to increase or replace such proteins with proteins produced by transformed microorganisms when the ability of a higher organism to produce such proteins is deficient and, for example, a symbiotic relationship between the microorganism synthesized by this invention and between a human with a chronic or acute deficiency disease, wherein the microorganism genetically transformed as described herein would be inoculated or otherwise associated with the human to compensate for a pathological deficiency in human metabolism.

As examples of specific DNA sequences of the present 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 activity is virtually eliminated. The intact mRNA is purified from the extract by column chromatography and exposed to the 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 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 single strand-specific nuclease that cleaves the simple linker strand. The resulting double-stranded cDNA is extended by adding to each end a specific DNA containing the restriction enzyme recognition site sequence. Appendix 15 is catalyzed by DNA ligase enzyme. 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 with the same restriction endonuclease recognition site is treated with the enzyme to cleave the polynucleotide strand and 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 trans-25-forming ring structure in 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 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.

The different steps of the method can be divided into four groups.

10 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 National Institutes of Health of the United States, human genes, whatever they may be, can be inserted into recombinant DNA and then into bacteria only if the genes have been thoroughly purified, 20 or if a particularly high-risk gene is used. working spaces (P4), see Federal Register, Vol. 41, no. 131, 1967-07-07, pp. 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 that are responsible for producing some of the protein in question. Alternatively, such a cell may be present at some stage in the development of the organism. Most of the mRNA isolated from the cells of such a cell contains the desired nucleotide sequence. Thus, the choice of cell to be isolated and method of isolation can take into account the advantages offered by the initial degree of purity of the mRNA to be isolated.

In most tissues, glands, and organs, the cells are joined by a fibrous connective tissue composed primarily of collagen, but may also contain other structural proteins, polysaccharides, and rock-like 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. The 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 other types of tissue cells.

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

In isolating rat growth hormone mRNA, treatment of cultured rat pituitary cells with thyroid hormone and glucocorticoids synergistically significantly increased 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 microorganism. As mentioned above, the RN donkey is widespread and is very active and exceptionally stable. It is on the skin, it withstands ordinary-35 glassware washing methods and sometimes it contaminates with organic chemicals.

14,74732 The present invention uses a combination of a chaotropic anion, a chaotropic cation, and a disulfide cleavage reagent during cell disruption and during all operations required to provide virtually protein-free RNA.

The choice of suitable chaotropic ions depends on their water solubility and availability. Suitable chaotropic cations include e.g. guanidinium, carbamoylguanidium, guanylguanidinium, lithium, etc. Suitable chaotropic anions include e.g. iodide, perchlorate, thiocyanate, diiodic salicylate, etc. The relative effectiveness of the salts formed by such anions and cations is determined by their solubility. For example, lithium diiodisalicylate is a more potent denaturant than guanidinium thio-15 cyanate, but has a solubility of only about 0.1 mol / L 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 5-mercaptoethanol, several suitable thiol compounds are known, such as dithiothreitol, cysteine, propanol dimercaptan and the like.

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 virtually complete, β-mercaptoethanol must be preferred because it is readily available at a reasonable cost.

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 mRNA intact during extraction is thought to be due to the rate at which the RNase, 5,74732, 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 defined as the threshold concentration required for complete denaturation of the protein. On the other hand, the rate of denaturation of proteins often depends on the ratio of the denaturant concentration to a threshold value of 5-10 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 hydrochloride can 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 preferable than lithium diiododisalate 20, although the latter is a stronger denatured bead 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, which is a slightly more potent denaturant, is more preferred than a nearly equally soluble 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 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, i.e., 74732, but it is preferred to use a large excess of thiol groups over intramolecular disulfide bonds to drive the exchange reaction toward the cleavage of intramolecular di-sulfide bonds. On the other hand, 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 8-mercaptoethanol is used to separate intact RNA, concentrations in the range of 0.05-1.0 mol / L have been found to be effective, and the optimal concentration is considered to be 0.2 mol / L.

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 is ethanol precipitation, with which RNA is selectively precipitated. In the process of the present invention, it is more preferable to omit the precipitation step and deposit the homogenate directly in a 5.7 mol / L cesium chloride solution in a centrifuge tube and then perform centrifugation in Glisin, V., Crkvenjakov, R. and Buys, C., Biochemistry 12, 2633 (1974). as described. This method is advantageous because the environment harmful to RN-25ase 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 the desired mRNA is present in this RNA. Further purification takes advantage of the fact that in cells of higher organisms, mRNA is processed after transcription by the addition of polyadenyl acid. Such mRNA containing poly-A sequences can be selectively separated on a chromatography column packed with cellulose to which oligothymidylate has been added, cf. Avis, H. and Leder, P., supra. The 17,74732 method described above is suitable when virtually pure, undamaged and translatable mRNA from RNase-rich sources is required. Purification of the mRNA and subsequent in vitro procedures can be performed in virtually the same manner 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 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 ent-20 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 by using mRNA as a template and a mixture of four deoxynucleoside triphosphates as a precursor of the DNA strand. It is preferred that one of the deoxynucleoside triphosphates is labeled at the 32 α 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, 30 A., et al., Supra.

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

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

Once the cDNA has been synthesized enzymatically, the RNA temp-5 plate 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 highly selective and can be 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 32 are as previously described, including α-β-labeled nucleoside triphosphate. Reverse transcriptase is obtained from several sources. Avian Myeloblastosis virus is a preferred source. The virus is manufactured by Dr. D.J.

20 Beard, Life Sciences Incorporated, St. Petersburg, Florida,

By 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 is preferred to use phenol extraction, chromatography ("SephadeiPb-100") and ethanol precipitation when DNA is to be purified from protein impurities.

The hairpin structure can be converted to a standard double-stranded DNA structure by removing a single strand connecting the complementary strands. There are several enzymes capable of specifically hydrolyzing single-stranded portions of DNA. A well-suited enzyme for this purpose is S1 nuclease isolated from Aspergillus oryzae (manufactured by Miles Research Products, 35 Elkhart, Indiana). When the DNA hairpin structure is treated with S1 nuclease, molecules with base pairs are obtained in good yield. After this executed 19 7473? Extraction, chromatography and ethanol precipitation are 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 of 10-10 acts so that the protruding 31-end is removed and the protruding 5 'end is filled. This ensures that as many cDNAs as possible are involved in the next step splicing reactions.

In the next step sa 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 inserted at the ends is selected on the basis of the restriction endonuclease and this 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 the Haemophilus aegyptious organism is purified by the following method: Middleton, J.H., Edgell, M.H. and Hutchinson III, C.A., J. Virol. 10, 42 (1972). The Haemophilus suis enzyme, Hsu I, catalyzes the same site-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 20,74732 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. above. Several such recognition site sequences are available to those skilled in the art, and it is therefore possible to select the ends of the double DNA to suit the restriction endonuclease required in each case.

The insertion of restriction site sequences into the ends of the cDNA can be accomplished by a method called tylp-10 end-to-end and 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 product cDNA having 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.

4. Construction of a Recombinant DNA Transfer Vector In principle, a variety of viral and plasmid DNA molecules could be used to generate recombinant and cDNA prepared as just described. The main requirements are that the DNA transfer vector be able to enter and replicate there in the host cell, and the vector should have a genetic trait that can be used to distinguish 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 according to NIH guidelines, cf. above. Suitable transfer vectors currently approved for use include e.g. 35 several bacteriophage lambda derivatives (see e.g. Blattner, FR, Williams, BG, Blechl, AE, Denniston-Thompson, K., Faber, HE, Furlong, LA, Grunwald, DJ, Kiefer, 21 74732 DO, Moore, DD, Schumm , JW, Sheldon, EL and Smithies, 0., Science 196, 161 (1977) and col E1 plasmid derivatives (see e.g. Rodriquez, RL, Bolivar, S., Goodman, HM, Boyer, HW and Betlach, MN, The ICN-UCLA Symposium is 5 Molecular Mechanisms In the Control of Gene Expression, DP Nierlich, WJ Rutter, CF Fox, Eds. (Academic Press, NY, 1976) pp. 471-477). relatively small size, in the order of a few million molecular weights, and the fact that the number of copies of plasmid DNA per host cell under normal conditions is 20-40, but can be increased to a thousand or more when the host cells are treated with chloramphenicol The ability of a host cell to increase the number of genes it contains will, under certain conditions, possible for the host cell to produce mainly 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 pBR313, pBR315, pBR316, pBR317 and pBR322, which contain the tetracycline resistance gene and the ampicillin resistance gene. The presence of the resistance-causing gene provides a suitable means of selecting cells 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 included in this description, a plasmid derived from col E1 containing the above resistance gene and one Hind 30 III recognition site was used.

Plasmid pBR322 has been highly characterized.

Bolivar, F. et al. (Gene 2 (1977) 95) has described the synthesis and characterization of this plasmid. Plasmid pBR322 has a molecular weight of 2.7 x 10 daltons (Bolivar, F., 35 Gene 4 (1978) 121) and contains ampicillin-resistant

R R

sin (Ap tn) and tetracycline resistance (Te) genes. The aforementioned Aβ gene has a single 22 7 4 7 3 2 £ 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 RI recognition site, two Hind II recognition sites, 5 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. Identification sites are indicated on the circular plasmid pBR322 on page 103 by Bolivar et al. In. The Aβ gene is lost when loh-10 is ligated from the plasmid Pst I recognition site and foreign DNA is inserted into it. Similarly, Te is lost upon cleavage of pBR322 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, recombination molecules are formed that can be detected from strains c that are both ampicillin-sensitive (Aβ) and tetracycline-resistant (Te) as well as by ligating DNA to the Hind III Sai I or Bam HI recognition site. recombination molecules are formed that can be detected in strains that are both ampicillin-resistant and tetracycline-sensitive. A transfer vector containing foreign DNA fused to one of the four recognition sites mentioned above can be characterized by the drug resistance minorities of the transfer vector, i.e., whether it is ampicillin-sensitive and tetracycline-resistant or ampicillin-resistant and tetracycline-sensitive. The transfer vector can be further characterized by removing the foreign DNA attached to it, determining the molecular weights of the two products formed, and comparing these to the molecular weights of the starting materials. In addition, the characterization can be supplemented by labeling the transfer sites of different endo-nucleases in the transfer vector. The sequence of the transferred DNA molecule can also be determined. The entire nucleotide sequence of said plasmid pBR322 is shown in J. Sutcliffe's dissertation-35 "Nucleotide Sequence of pBR322", Harvard University, Cambridge, Massachusetts, USA.

23 74732

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 4 daltons and contains 5 genes for ampicillin resistance (Aβ) and tetracycline resistance (Te). Plasmid pBR313 £

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

The third highly characterized plasmid is pMB9. This plasmid preparation method is described by Rodriguez, R.L. et al. (above) and Bolivar, F. et al. (Gene 2 (1977) 75). Plasmid pBM9 has a molecular weight of 3.5

x 10 ^ daltons and contains tetracycline resistance R

25 (Te) causing gene. This plasmid contains one recognition site for each of the following endonucleases:

Eco Rl, Hind III, Sal I and Bam HI. Of these three

O

recognition sites are located in the Te genes. When foreign DNA is ligated to the 30 recognition sites of Hind III, Sai I or Bam HI, 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.

24 74732

Plasmid pSC10I has been highly characterized. Cohen, S.H. et al. (Proc. Natl. Acad. Sci. USA 70 (1973) 1293) have described the synthesis and preliminary characterization of this plasmid. Characterization has been supplemented by Cohen, 5 S.N. et al. (Proc. Natl. Acad. Sci. USA 70 (1973) 3240,

Boyer, H.W. et al. (Recombinant Molecules), Beers, R.F. and Basset, E.G., Raven Press, New York, p. 12 (1977) and Cohen, S.N. et al. (Recombinat Molecules, p. 91). Plasmid pSC101 has a molecular weight of 5.8 x 10 ° daltons and contains the gene that causes tetracycline resistance (Te).

t>

The Te gene contains one recognition site for each of the following endonucleases: Hind III, Sal I and Bam HI. In addition, plasmid pSd01 contains one Eco RI recognition site, one Hpa I recognition site, one Sma I recognition site, and four Hinc II recognition sites. Tetra-cyclin resistance disappears upon cleavage of pSC101 by Hind III, Sal I or Bam HI endonucleases and insertion of foreign DNA into their recognition site. When ligated to the Hind III, Sai I, or Bam HI recognition site, re-20 combination molecules are found in cultures that are tetracycline sensitive. A transfer vector containing foreign DNA ligated to one of the three sites mentioned above can be characterized 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) constructing a recognition site map, and (c) determining the sequence of the transferred DNA molecule.

Several vectors can be used to transform Bacillus subtilis. These vectors are derived from the microorganism Staphylococcus aureus (see Fordanescu, S., J. Bakteriol. 124 (1974) 597 and Ehrlich, S.D., 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 HindIII site, and has a molecular weight of 1.8 x 10 3 daltons. The foreign DNA thus binds to the Hind III site of plasmid pC194. A transfer vector containing a 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.

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 when using P2 working facilities, has been developed for the methods described in this specification. Curtiss III, R., Ann. Rev. Microbiol. 30, 507 (1976). E. coli RR-1 is suitable when P3 working spaces are available.

Recombinant plasmids are constructed by mixing restriction endonuclease-treated plasmid DNA with cDNA terminated accordingly. Plasmid DNA is used in a large molar excess over the 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 recombinant insertion divides the gene and at the same time the function encoded by the gene is lost.

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-35a-digested plasmid DNA with alkaline phosphatase (manufactured by Worthington Biochemical Corporation, Freehold, New Jersey). Alkaline phosphatase removes phosphates from the 5 'to 26,74732 ends from the ends of the plasmid generated by the endonuclease and prevents self-ligation of the plasmid DNA. Thus, ring formation and thus transformation depends on the incorporation of a DNA fragment containing 5'-phosphorylated ends. The method described reduces the relative incidence of transformation without recombination to an amount less than 1-10.

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.

15 Table 1

Case Reactive compounds_Ligase product_ I 3 '5' 3 '5' -OH H -Ο-, ΡΟ - -OPO --- + Δ J + ou n -0P03H2 HO --- OPO- 20 5 '3' 5 '3' II 3 '5' 3 '5' - OH Η, Ο., Ρ-0- -OPO-

+ z J + H O

-OH OH --- OH HO - 2 25 5 '3' 5 '3' i III 3 '5' -OH HO- no reaction - OH + HO ---- 30 5 '3'

In Table 1, double-stranded DNA is shown schematically as solid parallel lines and their respective 5 'and 3' end groups are labeled hydroxyl (OH) or phosphates (OPOgH2). In case I, each of the reacting molecules contains 5'-phosphate at the ends, and thus the two strands covalently join each other 27 7 4 7 3 2. In case II, only one of the ends to be joined contains 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 telegram. The covalently unjointed strand remains associated with the joined telegram 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 compound reaction occurs.

10 It is therefore necessary to remove the 5 'phosphate group from the end to be prevented from joining the other. 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. U.S.

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

Reconstruction of the original sequence from the subfragments obtained by restriction 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 35 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 28,74732 is the preferred enzyme. 5 'terminal phosphates are a structural prerequisite for the binding activity of DNA ligase subfragments. Thus, ends without a 5'-terminal phosphate cannot be covalently joined. DNA subfragments can only be joined to ends that contain a 51-terminal phosphate formed as a result of the cleavage effect of the restriction endonuclease on the isolated DNA.

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, as 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. After high replication in E. coli, 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 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 report describes new microorganisms 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 novel microorganisms containing said genes.

29 74732

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, safety regulations do not require the isolation of cDNA 10 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 known to correspond to the known amino acid sequence of rat growth hormone. Cultured rat pituitary cells, a GH-1 subclone of the cell line and sold by the American Type Culture Collection, were used as a source of rat growth hormone 15 mRNA. Tashjian, A.H., et al., Endochrinology 82, 342 (1968). When such cells are grown under normal conditions, growth hormone mRNA 20 represents only a small percentage, i.e., about 1-3% of the total poly-A containing RNA. However, the level of growth hormone mRNA could be increased by the synergistic effect of thyroid hormones and glucocorticoids. RNA 0 was obtained from 5 x 10 cells grown in suspension-25 culture supplemented with 1 mM dexamethasone and 10 nM L-triiodothyronine 4 days before cell recovery. Polyadenylated 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.

30 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 (see examples of the parent application). Gel electrophoresis fractionation showed a weak but still clear band corresponding to about 800 base pairs of DNA.

30 7 4 7 3 2

Treatment of cDNA transcribed from the mRNA of whole cultured pituitary cells with Hha I endonuclease yielded two larger DNA fragments expressed by electrophoretic separation, corresponding to approximately 320 nucleotides-5 dia (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 strain application, it was found that these fragments were indeed parts of the rat growth hormone code when compared to published 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., Atlas of Protein Sequence and Structure, 15 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 similar 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 treatment, it was necessary to process the DNA to remove the odd single-stranded ends. Removal of such unpaired ends occurred prior to electrophoretic separation in a solution containing 25 μΐ 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 μΜ each). When the mixture was incubated for 10 minutes at 10 ° C with 1 unit of E. coli DNA polymerase I, the protruding 3 'ends were removed and the protruding 5' ends were filled exonucleolytically. Boehringer-Mannheim Biochemicals, Indianapolis, Indiana, sells DNA polymerase I.

A rat growth hormone containing about 800 base pairs was attached to a HindIII recognition site as described in Example 3 of the parent application. Plasmid pBR322, which had an ampicillin resistance gene and a Hind III site in the tetracycline resistance gene, was treated with Hind β endonuclease and alkaline phosphatase as described in Example 4 of the parent application. An 800-bp rat growth hormone cDNA was ligated to the treated plasmid using DNA ligase as 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 were grown on ampicillin-containing medium but not on medium containing 20 μg / ml tetracycline. Ten colonies were obtained with a plasmid joined by an approximately 800 base pair fragment cleaved by HindIII cleavage.

A preparative amount of 800 bp 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 parent application. The nucleotide sequence found contained at the 5 'end a region in which 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 isolation. 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) for positions 1-43, 65-69, 108-113, 133-143, and 150-190.

32 74732 du au 3 o c o wo wo HU h 3 h ait- 43 π - e>, <e 43 o o. -Jo p-jo a.:— oo n e n e H <e

<3 30 CO WO 30 0 C-O 30 O

. , '"J 0)! -> He> .0 - t— 3 H W <H C t-

g'H HO OO OH H C HO C O OO O

Q »D WO WO 30 30 W <H H 3 <Ö p £ <43 H 3 H >> C <» —I o o

. -g £ U H = U H H OO H C HH CO O

5 5¾ 30 030 3H 030 30 OHO WO <

T} 'dp 3 H (NOH H O CO 3 H H C -3-00;> -, <H

ό E> -1 u no e o no o o - h e n e h

Q Q 30 wo OH 30 Π H C C WO O

- -P 34 O H · —I C i — 1 O 3 H>, C - 1 c H C O

0_gc HO so <0 HU H H oo c: o o

^ 4Φ0 1-10 CO CO HO HO HO WO 30 O

Q + * 3 o h e - e ao 3 h> i <; 3 h H

34 w E- · oo <oo 00 n h> 0 n <00 0 P e

CHtÖ 30 30 30 30 600 30 C-O O

'd P W 43 t- HU - U 43 H H O 3 H W <O

C> m HH CO CO Π H CO OO CO o

E: P 0 | HU 60 C C H CO O 0.0 30 WO O

H -H g HO t · O WC h O W <i — i H> -> C H

Q Η β HC CO CC CO CO MC HC O

"p 34 _ DO 30 CO 3 H HO CO WO O

>> - | (0 3H 3 H i — I C 3 H 30 H <> iC- H

WJU + J HO HO OO HO w H OO HC O

(0 E 4-1 H

Ai Γ S 3 0 HO 3 H DO HO HO 30 O

WE 3 H 3 Η HH 3 Η 43 U HO HH O

C3 HO> U HC HH HC OO HH H

IJ fi i o

4J -H rn 0-0 3H HO 3 C HH 3H WO C

SHE ho HO OO H <HO HH>, O O

Sra HH CO W H OO OO HC OH C

W O

: ιβ · OO CH HH HO 3 H OOH HO C

: (6 (ö C ho w <>, e 3H hh ho 30 o

HCO HO CC HH SC HH CO W H O

H -P

: (0 3-1-) HH 30 COO HO HO OO 30 O -

Ifl 4-1 (0 400 ho HO W <3 H HO 3 H CO

-H-HW HC CO CO CO SC HO HO H

woe I

πω CO 3 H OCO HH 030 HO 030 30 I

njm> H <43 H c- ι-H e HO OOH 30 \ 0 3 H HH I

, g g Λί OO HH OO HC —ΠΟ WC - HO HH

• n C M H H DO HC CD C HO HO HO 3HC

rsl I 30 30 HO-HO 30 HO HO HO - '

-CC W H HO O O CC toilet OO OO CO H

- -Η (D Z H

O (0-HK HO HH 30 CO CO 0.0 HH WHO

34 W -P 6 WC 30 HC H <W <WC HC HO H

X CO W <00.0 0 CC CO HH OH

P> H> ® <HU OO CO HO DC CO HOO

H & frt 'ju 30 HO H <43 U HC wc 3 0 H

Pq3: (ö (3 e u wh ho oo hc oo e c wee

idW4J-P_, H

H-HWW 3 H DO 3 H 30 3 H DO WC HOC

Ό: (0 (Ο “<p ^ P h H HO HH 3H HC 3 0 O

H: (Ö> C U Hh hc CO HH HU HC WC H

OO · '0' - '^ O U HO DO 30 DO DU DC O

(fi C HO ^ ^ f "" HU H C HC HH H <3H HC O

H-3-H'SC HO HH OO HC OO HO OOH

x ε w c 3raW π p 30 DO 600 CO 3 0 3 0 0

CC. 3 H HO HC HO H <3 H HOH

!>, © - <<u oo cc oo ho eo <;

& 4J X 30 00 H wo HO HO 3H 3 H H

3 ---) 3 HO HO H <30 3 H HU H H H

35 CO CO H C WC SC CO HHC

§ s e ° H DO H O DO DO 0.0 OO U C

Ss-P HO hc HO 3 H 3 H WC HOC

^ ÖC H U OO OO HO HO CO COH

i | 0 § 8 - DC 30 HO 3 H 3 H HH OOU <!

rX n 3 H 4 = H 430 HH HO WC HOH

HO H H HC HH CO CO COO

§ 8.3 u «H DO O O O CO OCO HU OWH H

frtJrtS O HO - e OHO HC CMHC 30 00> - O O

-C-C (0 U CO OO HO OO - OU toilet - OH H

"S-Ti! Rt u H

Η >> H WC 30 HO 30 00 O WOH

C-P15 P —'O >> C - O 3 H Ht- HO * C U

c oo Η-ί cu> o hc co hcc

(oO. f- 3H HU OC Ο Ο >> U H O Hl O C

{Xjj-j O HO> .e HO HO HO 3 H 3 H H

UliöfO <C O HH HO HO OU SC SC O

8 s Λ ti 3U HU 30 >> 0 DO CU HOO

D 3 Π <H C 430 HH HO HC WC 3HO

H + JW ^ UU HC HC OU OU CC> OC

SmiS y c C HO HU DU DC 30 6000

(¾¾¾ <r4-4 wc 430 3 H HC HU HOO

^ ^ w o oo CO HC HU OO CO COH

P CJ

pH H Cu o DO CL υ DO <V ~ ° r-IO rH <μ O Q) H w <0> H <

HU <u OO HH HO <0 HOO

I o

J p-p 3 H H C H c HO 3 H HOO

U. HP. "Ί 0 HU 3 O WC 43 t->, CH

HH CO WH WH CO Hh HHU

33 74732

Example 2a (i) Example 1 was repeated except that the DNA of plasmid pBR322 was replaced by DNA of plasmid pMB9 (method of preparation: Rodriguez, RL, Bolivar, F., Goodman, 5 HM, Boyer, HW and Betlach, M., ICN-UCLA symposium on

Molecular and Cellular Biology, D.P. Wierlich, W.J. Rutter and C.F. Fox, Eds., (Academic Press, New York 1976), p. 471-477). The conditions used were otherwise the same as in Example 1, except that the selection was performed as in Example 4. 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 pSC101 DNA prepared by 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 pMB9. 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 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 2b

Examples 1 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.

34 74732

Example 3 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 2, 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 mol / l guanidinium thiocyanate. and 1 mol / L mercaptoethanol and buffered to pH 5. The homogenate was layered on 1.2 ml of 5.7 mol / L CsCl containing 100 mM EDTA and centrifuged for 18 hours at 15 ° C at 37,000 rpm. Beckman ultracentrifuge with 20 rotors SW 50.1 (Beckman Instrument 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 of the parent application. Approximately 10% of the 25 RNAs thus isolated encoded growth hormone as judged by the incorporation of a reactive amino acid precursor 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-35 polymerase I as described in Example 2 and then attached to Hind β recognition sites. The cDNA was then recombined with DNA ligase into 35 7 4 7 3 2 plasmid pBR322 / treated with alkaline phosphatase. E. coli X-1776 was transformed with recombinant DNA and a strain containing growth hormone DNA was selected. The strain containing growth hormone DNA was grown in pre-5 parity and growth hormone DNA was isolated for nucleotide sequencing. The cloned growth hormone DNA 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 HjN-Phe-10 Pro-Thr-Ile-Pro-Leu-Ser-Arg-Leu-P? IE-Asp-Asn-Ala-Met-Leu-

Arg-Ala-His-Arg-IIIS-His-Gln-Leu, The remainder of the sequence is shown in Table 3.

36 74732 .J · 3 0 HU HU 3 0 M S- • 5 _ OH >> <w <O h (J ϋ

C W -1 ^ e— H <O m3 U in H

n W U 3 U - O 3 mi f 1

g G OH 0 5 — t r-4 <3 o -— M CJ

Q S g o _iu> α o o ju <o B M <- o 3 o 3 cj 30 ra <; wo 0 <U _ - <3 OH OH> -4 CJ >> o

X Ö) CO OO, -30 —3 u <o o H

H I v) <j

!> <<< 3 <OCO COO HO CO

tog _ 05- C W <! MO W <- <<

jö ^ - · << <<<o OO

OO CO co> * o WE- o—> o

SaJ w <m <3 o · - <oooc- (3 30 <3 <<3 <OO so -> o W 43 O H n

• H 10 - o MO OO 4-1 O CO <DO

E {0 no -1O OH W <; m4 H I

£> CJ O C / 3 H <o X <<3 <J M <I

• H H H W <CJU 30 WO COO!

mtn, - 33 5- OH H <MO I

(ti W ^ it <h <3 Π H H o so <o

a g rO O O CJ

P δ Ί W r- CO r-io MO M <3 30 O

cd>., * -> <Ο Η -CO OO OH o M t H c u> o H <C / 3 H H o o

4-> 0) He- C <MH C <CO ΟΌ O

cl -! <oo »-, <w <; s h o

• H <C “> ^ o O C / 3 <OO <<π H H

O z h <; cj

HO i-3 <M <iS U 0) U M <M <3 H

EC 30 H <3 <3 <3 H <3 Η <H <3 O

g-H O f— <Cj ZC · <ΓθΟ 3 <30 O 3 »U Π O 30 c-

Q <- <OH CN U W <m4 <O

.g 3 <OO m3 O - OO <0 OO <

H C - -C CJ

m o OO 30 OO 3 <OO r-o O

tn_2 - <S3— H <s H CH o c "9 JP U t— H h o o Π H m- o o 5> ω m3 ^ eco co so wo wo o

& frt - WO - <--1 <3M <? -, <O

aj ΰ o <c <o o oo o <s <ο * Λ mo cj

Jj e HO CO r-to 3 <MO HO <

rHffl W <CH OH OO mc O

.y g OO <<> 0 m3 o m <<O H

n I — 1 <MO OO HU MU 4-1 CJ H

n M 5 o o mo w <> s <o— o

iiJ a MH in H HO <0 HH Ο <O

0-¾. HH OO 3 0 WO MO HO H

λ; 2 pf m o r- <>> <so me <

A3 C "tf 30 HO OO -3 <H <<0 O

e r- · m -J '—i U O

P4Mrico <^ Μ <30 3 <CO wo o

'O} H + J O S O OH OH - <H <H

2Si'fr 3 <OH <m3 O m3 O OO -3 <O

H: d W O O O O HO 3 0 O W O CO O O

• S MO MO OH -T ΪΜ <MO O

Ψ. J 3 <HO HH m3 O - m3 <<< O

JL 4 — I -S O

S-H OO OH M O HO OO OO O

S.2J r — t H OO W <JZ H JZ H H

> -3.5 OH M <tn H <O HH HH O

Sfr HH MH CO HH OO WO O

Q OO i— <>, <> - · H>% o <

y R 3 0 ¢ / 3 H OO HH> - <OH H

• R g OCJ 30 OU m-ι CJ CO HU · —1 O O

.to · r—, -tt HH OH r-i <3 H e C'SH

C (tJ CO OO H <> U OU HH · - · H H

9 C, H H <

g-r ^ rH OU H <30 CO >> U 30 >> <J

Q W ·· <UO D ►— <Cfl <! <U £ - * H C

Ewd HU in i— m3 O <3 <OO m3 o oo

QCpC HH OO 30 HO HH 30 WH

-S> <2 JC H OH OO -CO OH? S O

S'SjJ mU HH m3U W <H <m3 O OH

WW [dH <WH 30 HU 600> iO HU

(tJQ> >> U OOH WC HO 1-4 O OO

.Y Cl, H HO OH CO m3 O <U <0 OO ¢ / 3 <SO w <

S (Ö, Y <U 30 HU HH OO OHO >> O

X 4) t- OU OO HO vO >> <· —4 O

U) OE-4 O'- m3 O OH WH HO - HH

E-HCHH HU OU «Ο HO CO 30 -CEp OU H M-iCJ OO W <C“! <

H B Ä 30 V3H I— <<O m <<<, OO

<- υ

N <o MO 600 >> 0>% 0 WO -4 CJ

SO MO —4 O —40 J? '- 1

CJ HC <CJ OO OO m3 <> U

in 37 747 3 2

Example 4a (i) Example 3 was repeated except that plasmid pBR322 DNA was replaced with plasmid pMB9 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 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 bp and having the nucleotide sequence shown in Example 1.

(ii) Example 3 was repeated except that plasmid pBR322 DNA was replaced with plasmid pSC101 DNA prepared by Cohen et al. by the method described in (Proc. Natl. Acad. Sci. USA 70 (1973) 1293).

The conditions used were the same, and selection was performed in the same manner as when using plasmid pMB9. 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 3.

(iii) Example 3 was repeated except that plasmid pBR322 was replaced with plasmid pBR313 DNA prepared by Bolivar et al. by the method of 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 3.

Example 4b

Examples 3 and 4a 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.

Claims (3)

A DNA transfer vector which contains a nucleotide sequence encoding growth hormone and which can be used in transforming a microorganism, characterized in that the transfer vector contains a deoxynucleotide sequence encoding human growth hormone which is as follows: - 'ttk1ccl2acl3atm4ccl5x6ty6qr7s7w8gz8x9ty9ttk10gak11aak12 GCL, 3ATGi 4X, styrene, 5W, ggz, gGCL, AK ^, ^, 9X20TY20CAK21CAJ22X23TY23 GCL24TTK25GAK26ACL27TAK28CAJ29GAJ30TTK31GAJ32GAJ33GCL34TAK35A ™ 36 CCL37AAJ38GAJ39CAJ40AAJ41TAK42QR43S43TTK44X45TY45CAJ46AAK47CCL48 15 CAJ49ACL50QR51S51X52TY52TGK53TTK54QR55S55GAJ56QR57S57A ™ 58 CCL59ACL60CCl «1Q ^ * 2S62AAK63W64G 64GM« 5GM66, KCl, 67CAJ6eCAJ69AAJ70 QR71S71AAK72X73TY73GAJ74X75TY75X76TY76W77GZ77A ™ 78QR79S79X80TY80X81 20 TY81X82TY82A ™ 83CAJ84QR85S85TGG86X87TY87GAJ88CCL89GTL90CAJ91 TTK92X93TY93W94GZ94QR95S95GTL96TTK97GCL98AAK99OR100S100X101TY101 GTL102TAK103GCL104GCL105QR106S106OAK107QR108S10eAAK109GTL110 CEILING) in iGAKi in 3TY11 2x11 3x11 6x11 4TY11 4AAJ11 5GAK11 7TY11 7GAJ118GAJ11 9 25 GGL120A ™ 121CAJ122ACL123X124TY124ATG125GGL1 26 W1 27GZ1 2 7x1 2BTY1 28 GAJ129GAK130GGL131QR132S132CCL133W134GZ134ACL135GGL136CAJ137 A ™ 1 38TTK1 39AAJ1 40CAJ1 41ACL142TAK143QR1 44S144AAJ1 45TTK1 46 GAK147ACL148AAK149QR150S150CAK151AAK152GAK153GAK154GCI'155 30 X156TY156X157TY157AAJ158 AAK159TAK160GGL161X162TY162X163TY163TAK164 TGK165TTK166W167GZ167AAJ168GAK169ATG170GAK171AAJ172GTL173GAJ174 ACL1 75TTK1 76X1 77TY1 77W1 78GZ1 78A ™ 1 79GTL1 80CAJ181TGK1 82W1 83 25 GZ183QR184S184GTL185GAJ186GGL187QR188S188TGK189GGL190 TTK1 91 --- 3 where 42 7 4 7 3 2 A is deoxyadenyl, G is deoxyganyl, C is deoxycytocyl, T is thymidyl, J is A or G, K is T or C, L is A, T, C or G, M is A, C or T, X_ is T or C, if Y is A or G, and C, if Y is nnn 10 is C or T, Yn is A, G, C or T, if Xn is C, and A or G, if X is T, n W is C or A, if Z is G or A. and C, if Z nnn is C or T, Zr is A, G, C or T, if V is C, and A or G, if W is A, n QRn is TC, if is A, G, C or T, and AG, if S is T or C, n S is A, G, C or T if QR is TC, and T or C, nn if QR is AG, n and subheading n denotes the position of the amino acid in human growth hormone corresponding to the nucleotide sequence, according to the genetic code, and the amino acids have been numbered from the amino end. Vector according to claim 1, characterized in that J is A in the following positions: 32, 33, 39, 66, 68, 70, 119, 122 and 129, J is G in the following positions: 29, 30, 38 , 40, 41, 30 46, 49, 56, 65, 69, 74, 84, 88, 91, 115, 118, 137, 140, 141, 145, 158, 168, 172, 174, 181 and 186, K is T in the following positions: 25, 31, 35, 42, 53, 111, 130, 153 and 189, K is C in the following positions: 26, 28, 44, 47, 54, 35 63, 72, 92, 97, 99 , 103, 107, 109, 112, 116, 139, 143, 146, 147, 149, 151, 152, 154, 159, 160, 164, 165, 166, 169, 171, 176, 182 and 191,