SE455794B - PLASMID WITH NUCLEOTIDE SEQUENCE CODING THE AMINO ACID SEQUENCE IN HUMAN GROWTH HORMON AND PROCEDURE FOR PRODUCING THEREOF - Google Patents

Abstract

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

Description

455,794 corresponding DNA segments, containing a sequence of triplets, which corresponds to the amino acid sequence of the protein. The genetic code is shown in the table below.

The biological process, which involves the conversion of the information inherent in the nucleotide sequence into amino acid sequence structure, involves a first step, called "transcription".

In this step, a local DNA segment is first copied, with RNA, the sequence of which prescribes which protein is to be produced.

RNA is a polynucleotide, which is similar to DNA, however, in RNA ribose is included instead of deoxyribose and uracil is included instead of thymine.

The bases in RNA can form the same kind of base pairs as the bases in DNA. The RNA transcription product of a DNA nucleotide sequence therefore becomes complementary to the copied sequence. The RNA thus obtained is called messenger RNA (messenger RNA, mRNA), because it is an intermediate product between the cell's genetic apparatus and the protein synthesizer.

Within the cell, mRNA was used as a template in a complicated process; this process, which takes place with the help of many different enzymes and organelles within the cell, results in the synthesis of the specific amino acid sequence. This process is called "translation" of mRNA.

Often additional process steps are added, which are performed to convert the amino acid sequence synthesized by translation or translation into a functional protein.

Genetic code Phenylalanine (Pm) TTK Histiain (His) cAx Leucine (leu) XTY Glutamine (Gln) CAJ Isoleucine (Ile) ATM 'Asparagine (Asn) AAK Methionine (Met) ATG Lysine (Lys) AAJ Valine (Val) GTL Aspartic acid ( Asp) GÅK Serine (Ser) QRS Glutamic acid (Glu) GÅJ Proline (Pro) CCL Cysteine (Cys) TGK Treonin (Thr) ACL Tryptophan (Try) TGG 455 794 Alanine (Ala) GCL Arginine (Arg) wgz Tyrosine (Tyr) TAK Glycine (Gly) GGL Stop Signal TAJ Stop Signal TGA Note, Each triplet indicated by a three letter represents a trinucleotide in the DNA molecule, which trinucleotide on the left side has a 5 'end and on the right side has a 3' end. The letters indicate the purine or pyrimidine bases in the nucleotide sequence.

Many proteins, which are important in medicine or for research purposes, are found in or produced by the cells of higher organisms, such as vertebrates. These proteins include, for example, the hormone insulin, other peptide hormones, such as growth hormone, proteins that are active in regulating blood pressure, and various types of enzymes that are important in industry, medicine or research. It is often difficult to obtain such proteins in useful amounts by extraction from the organism; this problem is particularly acute in the case of proteins of human origin. There is therefore a need for technology to provide that such proteins in acceptable amounts can be produced by cells outside the organism in question. In some cases, suitable cell lines can be obtained, which can be kept alive by tissue culture technology. However, the cells grow only slowly in tissue culture, and the culture medium is expensive, the conditions must be carefully regulated, and the yields are low. In addition, in many cases it is difficult to maintain a cultured cell line which has desired, differentiated characteristics.

In the case of microorganisms - such as bacteria - on the other hand, they can be grown relatively easily in chemically defined media. The fermentation technology is a very advanced technology and can be controlled well. The organisms grow rapidly, and high yields can be obtained; In addition, certain microorganisms have been thoroughly genetically characterized; this to such an extent that they can be counted among the best characterized and best explored organisms.

It is therefore highly desirable that a gene, which is a code for a medically important protein, should be able to be transferred from an organism normally producing the protein to a suitable microorganism. On this sweet, the protein can then be produced by the 455 794 microorganism under controlled growth conditions and obtained in desired amounts. It is also possible that thanks to such a process the total cost of manufacturing the desired protein could be significantly reduced. In addition, the possibility of isolating the genetic sequence that determines the production of a particular protein and transferring that sequence to a micro-organism that has a well-defined genetic background must be considered as an extremely valuable asset for research purposes. to investigate, how the synthesis of such a protein is regulated and what further processes the protein undergoes after being synthesized. In addition, isolated genetic sequences can be altered to code for variant proteins, which exhibit altered therapeutic or functional properties.

The present invention makes it possible to achieve the above-mentioned objects. The invention thus relates to a plasmid suitable for transferring a gene to an Escherichia coli bacterium, and this plasmid is characterized in that it contains in its nucleotide sequence a sequence which encodes the amino acid sequence of human growth hormone and comprises 5 '- -TTR, CCL2ACL3ATM4CCL5X6TY6QR7S7W¿GZ5X9TY9TTK, OGAKIIAAKI2 GCL, 3ATG ,, X, STY SWIGGZ, 6GCL,; CAK, $ W, 9GZ19X20TY20CAK2, CAJ22X23 TY23GCL24TTK25GAK2GACLZ7TAR28CAJ29GAJ30TTK3, GAJ32GAJ33GCL3, TAK35ATM36CCL37AAJ38GAJ39CAJ4OAAJ4 roof 2QR43S43TTK44X45TY, 5 CAJ4sAÅK41CCL4aCAJ49ACLsoQRs15sjXs2TYs2TGKssTTKs4QRss3ssGA5se QRs15s1ATMssCCLseACLsoCCLs1QRG25e2ÅAKssWs4GZe4GAJssGÅJssÅCLs7 CAJssCAJssÅAJ1oQR71511AÅKv2X? aTYvaGÅJ14X1sTY1sX? eTYvsW11GZ17 ATM1sQR1s51sXsoTYsoXs1TYs1Xs2TYs2ATMaaCAJa4QRss5asTGGasXs1 TY ¿7GAJs¿CCL¿9GTL90CAJ9ITTKQQXQ3TY93W9, GZ94QR95S§5GTL96TTK91 GCLssAÅKeeQR1oo51ooX1o1TY1o1GTL1o2TAK1o3GGL1o4GCL1osQR1oe51os GO: o7QR1os5: osAAK1osGTL11oTAK111GAK112X1: sTY11aX :: 4TY114 ÅÅ311sGÅK11sX11ïTY111GÅ311sGAJ11eGGL: 2oATM: 21CÅJ122ACL12s X124TY 124ÅTG12sGGL12eW: 21GZ: 21X: 2eTY: 2sGAJ: 2sGÅK: soGGL1s: QR1s251a2CCL1ssW1s4GZ1a4ACL1ssGGL1ssCÅ3: a1ATW1asTTK1asAÅJ: 4o CA3141ÄCL142TÅK14sQR1445144AAJ14sTTK14eGAR141ACL: 4sÄÅK14s QR1so51soCAK1s1AÅR1s2GÅK1saGÅK: s4GCL1ssX: saTY1scX1s1TY1s1 A331ssAAK1seTAH1soGGL1e1X: e2TY1e2X1esTY1saTÅR1s4TGK1asTTK1sc W s1GZ1s1AA31esGAK1ssÅTG11oGÅK111AÅ3: v2GTL: 1sGÅJ1v4ÄCL1vs TTK11sX117TY177W17sGZ11sATN11eGTL1soCA31s1T5K1szW: ssGZ1sa QR1s451s4GTL1ssGAJssGGL1s1QR: SS5: asTGK1s9GGL1euTTR; f'3 ' * 5,455,794 where A is deoxyadenyl, G is deoxyguanyl, 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 when T or C '° m Yn is A or G, and C if Yn is C or T; Yn is A, G, C or T if Xn is C, and A or G if Xn is T; Wn is C or A, if Zn is G or A, and C if Zn is C or T; Zn is A, G, C or T, if Wn is C, and A or G if W 'is A; QRn is TC, if Sn is A, G, C or T, and AG if Sn is T or C; Sn is A, G, C or T, if the QRn is TC, and T or C if the QRn is AG and the index numbers, n, indicate the position of the amino acids in human growth hormone, for which the nucleotide sequence constitutes the equivalent according to the genetic code, wherein the amino acid positions are numbered based on the amino terminus.

The above sequence is correct and differs from that stated in claim 1 in that AAK100 has been corrected to QR100 S100 and CAK153 has been corrected to GAK153. The original inaccuracies are due to unintentional errors in the printing of the experimental results from the sequencing of the gene. A person skilled in the art would not have been misled by the incorrect information, since Bewly et.al. already in 1972 (International Journal of Peptide Research, 1972 (4), 281-287) indicated Ser / AGC in position 100 and Asp / GAT in position 153 for human growth hormone.

The above plasmid is prepared by a method comprising the steps of: α 455 794 (a) isolating mRNA from pituitary cells by homogenizing the pituitary cells in the presence of an RNase inhibitor composition containing 4M guanidinium thiocyanate od10, O & -1, 0M B-mercaptoethanol at pH 5.0-8.0, thereby preventing RNase degradation of this mRNA; (b) producing cDNA having a nucleotide sequence encoding growth hormone by treating recovered mRNA with a reverse transcriptase in a manner known per se, after which this CDNA is double-stranded; (c) attaching sequences with a restriction endonuclease recognition site to the ends of the resulting double-stranded cDNA, the restriction endonuclease being the same as in step (d) below, after which the resulting cDNA is cleaved with the restriction endonuclease to form cohesive ends; (d) a bacterial plasmid is opened with restriction endonuclease} eg Hind III or Hsu I, to form an opened plasmid vector with cohesive ends; (e) The 5 'phosphate end groups of the plasmid vector obtained in step (d) are hydrolyzed by treatment with alkaline phosphatase, for example at pH 8.0 and the temperature 65 ° C for 30 minutes, whereby those under (d ) said cohesive ends are rendered incapable of bonding together; and (f) the plasmid vector treated in step (e) is linked to the cDNA obtained in step (c) by incubation in the presence of DNA ligase and ATP, for example at pH 7.6 and the temperature 14 ° C for 1 hour using of a molar excess of the plasmid vector, thereby obtaining a plasmid having a nucleotide sequence encoding growth hormone.

The invention thus comprises i.a. a process with a complex series of steps involving enzyme-catalyzed reactions.

In the following, the principles of these enzyme reactions are described, as they are understood in accordance with the prior art.

Reverse transcriptase catalyzes the synthesis of DNA, which is complementary to a RNA strand serving as a template, which synthesis takes place in the presence of both the RNA template and a so-called "primer" (the starting directing molecule), which consists of an oligodeoxynucleotide, and the four deoxynucleoside triphosphates dATP. dGTP, dCTP, TTP. The reaction is initiated by binding the oligodeoxynucleotide molecule non-covalently to the 3 'end of the mRNA molecule; then m 1 455 794 follows stepwise addition of the "correct" deoxynucleotides to the 3 'end of the growing chain. i.e. addition of the deoxynucleotides which, in accordance with the base pairing rules, are the "correct" ones for the bases present in the current mRNA nucleotide sequence. The molecule thus obtained as a product can be described as a hairpin-shaped structure, containing the original RNA together with a complementary strand of DNA, which is linked to the original RNA by means of a DNA strand consisting of a single strand.

Reverse transcriptase can also catalyze a similar reaction using a template. which is a DNA consisting of a single strand; In this case, a hairpin-shaped DNA is obtained as a product which consists of two strands and has a loop of single-stranded DNA as a connection between the ends of the molecular parts. See Aviv. H. and Leder, P., Proc. Natl. Acad. Sci.USA 6Q¿ 1408 (1972), and Ef- stratiadis, A., Kafatos. F.C., Maxam: -ÄTF. and Maniatis. T .. Qgll 1. 219 <19v6>.

Hestriction endonucleases are enzymes capable of hydrolyzing phosphodiester bonds in two-stranded DNA, thereby disrupting the continuity of the DNA strand. If the DNA molecule is in the form of a closed ring or loop, it is transformed into a linear structure. What is particularly characteristic of an enzyme of this type is that its hydrolysing action takes place only at a site where a specific nucleotide sequence is present. Such a sequence is called a "recognition site" for the restriction endonuclease. Restriction endonucleases from various sources have been isolated and characterized, the characterization taking place in the form of identifying the nucleotide sequence of the recognition sites for these enzymes. Some restriction endonucleases hydrolyze the phosphodiester bonds on both strands at the same site, resulting in "dull cut" ends.

Others catalyze hydrolysis of bonds at mutually mutated sites, in that these sites are spaced apart by a few nucleotides. In this case, free single-stranded regions are formed at each end of the cleaved molecule. Such single-stranded ends are self-complementary and therefore cohesive; they can be used to reassemble the hydrolyzed DNA molecule. Because any DNA that is cleavable by such an enzyme must contain the same recognition site. the same cohesive ends will be formed; thereby making it possible for heterologous sequences of DNA which have been treated with restriction endonuclease to be linked to other sequences which have been similarly treated. See Roberts, R.J., Q§L§¿_§g! ¿_§12; 9g3m¿ Q, 12) (1976). Restriction sites are relatively rare, but the utility of restriction endonumases has been significantly increased by chemical synthesis of oligonucleotides. which consists of two strings and shows the sequence of the restriction site. Therefore, in fact, virtually any DNA segment can be linked to any other segment by simply binding an appropriate restriction oligonucleotide to the ends of the molecule and subjecting the product to the action of the corresponding restriction endonuclease, so that it is hydrolyzed by this endonuclease and thereby gives rise to the formation of the required ones. cohesive ends. See Heyneker, H.L., Shine. J., Goódman, HM, ßoyer, HW, Rosenberg, J., Dickerson, RE, Narang, SA,> Itakura, K., Lin, S. och Riggs, AD. Fl atugg g§§, THB (l976), samt Scheller, RH, D1ckerson, RE, Boyer, H, w., Fi iggs. A.D. and Itakura, K., §gigggg šgš, 177 (1977) - S1-endonuclease is an enzyme which has general specificity and is capable of hydrolyzing the phosphodiester bonds either by a single strand of DNA or by a single strand permanent gaps or loops in a DNA molecule, which otherwise consists of two strands. See Vogt, V.M., Eur. J. Biochem.} §, 192 (wmi. "_ _ - DNA ligase is an enzyme which is capable of catalyzing the formation of a phosphodiester bond between two DNA segments with a 5 'phosphate group and a 3' phosphate group, respectively. hydroxyl group, of the type which could be formed by two DNA fragments, which are joined together by cohesive ends.The normal function of the enzyme is assumed to be the bonding of short sections (nicks), consisting of a single strand, in an otherwise of two strands However, under suitable conditions, the DNA ligase is capable of catalyzing the bonding of "dull-cut" ends, whereby two molecules with such "dumb" ends are covalently linked, See Sgaramella, V., Van de Sande, JH and Khorana, HG Proc. Natl. Acad.Sci.USA, gg. 11162 (me).

Alkaline phosphatase is an enzyme that has general specificity and the ability to hydrolyze phosphate esters - including 5 'end-position-bound phosphates on DNA. 9, 455 794 Another step in the intended method 1 in its entirety is the insertion of a specific DNA fragment into a plasmid. Hed "plasmid" refers to an autonomously replicating DNA unit of any kind that may be present in a microbial cell, except for the host cell's own genome. A plasmid is not genetically linked to the chromosome of the host cell. Plasmid DNA molecules exist in the form of annular, two-stranded molecules with a molecular weight, which is usually of the order of a few million, although some have a molecular weight exceeding 10, and they usually form only one small percentage of the cell's total DHA. Plasmid DNA can usually be separated from host cell DNA because these different types of DNA molecules have such different sizes. Plasmids can undergo replication regardless of the rate at which host cell division occurs; in some cases, their rate of replication can be controlled by the researcher through variations in the growth conditions. Although the plasmid is in the form of a closed ring. is it possible to artificially insert a DNA segment into the plasmid; thereby forming a larger molecular size recombinant plasmid, without its ability to replicate or express the plasmid-borne genes being significantly affected. The plasmid is therefore valuable as a vector for transferring a DNA segment to a new host cell. Plasmids which are useful for the technology relating to recombinant DNA typically contain such agents which may be useful for selection purposes, e.g. genes for resistance to drugs or medicines. By obtaining DNA with a specific sequence, which constitutes the genetic code for a specific protein, it becomes possible to modify the nucleotide sequence chemically or biologically in such a way that even the specific protein finally formed is modified. This makes it possible to produce e.g. a modified growth hormone, which is "tailored" to a specific medical need. The genetic capacity to produce any growth hormone-related amino acid sequence with the most important functional properties of growth hormone can therefore be transferred to a microorganism. -455 794 10 Due to the fact that the genetic code for a specific protein, which is required for the normal metabolism of a certain particular higher organism, can be transferred to a microorganism, e.g. a bacterium, one gets great opportunities for culture production of such proteins. This in turn provides great opportunities to increase the production of such proteins or replace them with proteins produced by microorganisms altered according to the invention, whenever the ability of the higher organism to function normally in the production of such proteins has been impaired. In connection with this, one can also see the possibility that e.g. establish symbiosis relationships between microorganisms prepared according to the present invention and humans with chronic or acute deficiency diseases, whereby microorganisms which have been genetically modified in accordance with the present invention could be implanted in or otherwise associated with the patient in order to obtain compensation for the pathological deficiencies in the patient's metabolism.

More particularly, the invention relates to a method for isolating a specific nucleotide sequence, containing genetic information, synthesizing DNA with the specific nucleotide sequence, and transferring this DNA to a bacterial host host.

In the method according to the invention, a selected cell population is first isolated by means of an improved method. Intact mRNA is extracted from the cells by a new procedure, in which virtually all ribonuclease activity is suppressed. Intact mRNA is purified from the extract by column chromatography and subjected to the action of the enzyme reverse transcriptase, which acts in the presence of the four deoxynucleoside triphosphates needed to synthesize a complementary (cDNA) strand. The product of this first reaction with reverse transcriptase is subjected to a process in which the ribonucleotide sequence is selectively removed. The then remaining deoxynucleotide sequence, which is complementary to the original mRNA, is incubated in a second reaction with reverse transcriptase or DNA polymerase in the presence of the four deoxynucleoside triphosphates. The product. which is then obtained, is a double cDNA structure. whose complementary strands at one end are interconnected via a loop, consisting of a single string. This product is then treated with single-stranded specific nuclease. which splits it from a string - 455 794 11 consisting of the loop. The then-formed, two-stranded cDNA is then extended by adding at its two ends a specific DNA, containing a sequence which constitutes a recognition site for a restriction enzyme. The addition is catalyzed by a DNA ligase enzyme. Thereafter, the extended cDNA is treated with a restriction endonuclease, and in this way is formed at the end positions of each strand self-complementary, single-stranded nucleotide sequences.

A plasmid DNA, which has a recognition site for the same restriction endonuclease, is treated with this enzyme so that the polynucleotide strand is cleaved and nucleotide sequences are formed at the 5 'end positions. which consist of a single string and are self-complementary.

On the single stranded end portions, the phosphate groups present at the 5 'ends are removed to prevent the plasmid from forming a ring structure capable of converting a host cell. The prepared cDNA and the prepared plasmid DNA are incubated together in the presence of DNA ligase. Under the reaction conditions described, a viable closed ring of plasmid DNA can be formed only if a segment of cDNA is included. The plasmid, which contains the cDNA sequence, is then introduced into a suitable host cell. The cells which have received a viable plasmid can be recognized by the emergence of colonies with a genetic trait which they have acquired from the plasmid, e.g. resistance to a drug. Pure bacterial strains containing the recombinant plasmid with the cDNA sequence incorporated therein are then grown and the recombinant plasmid is again isolated. Large amounts of recombinant plasmid DNA can be prepared in this way, and the specific cDNA sequence can be re-isolated from this plasmid by endonucleolytic cleavage with the appropriate restriction enzyme.

The invention is described below in greater detail.

According to some embodiments of the invention, there is provided a method of isolating a DNA molecule having a specific nucleotide sequence and its transfer to a microorganism, the original nucleotide sequence of said DNA being present after replication in the organism to which said transfer has taken place.

The process according to the invention can be divided into four general steps. 455 794, 2 l. Isolation of a desired cell population from a higher organism.

There are two potential sources for a genetic sequence, which is the code for a specific protein: the DNA itself in the source of the selected organism, and an RNA transcript (RNA transcript) of this DNA. According to the f.n. According to US safety regulations, issued by the National Institutes of Health, human genes of any kind may be introduced into first recombinant DNA and then bacteria only after the genes have been very carefully purified or also in specially built for high-risk premises (P4). See Federal Register vol. ül, nr.'l3l, July 7, 1967, p. 27902-å794}.

In the case of processes with potential utility for the production of human protein, e.g. In the present method, it is therefore always preferred to go the route of isolating specific mRNA with a nucleotide sequence encoding the desired protein.

This approach also has the additional advantage that the mRNA in question can be purified more easily than DNA extracted from the cell.

In particular, it can be mentioned that one can take advantage of the fact that in the case of highly differentiated organisms, such as vertebrates, it may be possible to identify a specific population of cells with a specific site in the organism, the function of which is primarily is precisely the production of the protein in question. Alternatively, such a population may. exist during a transitional stage in the development of the organism. In such cell populations, a large portion of the mRNA isolated from the cells will have the desired nucleotide sequence; For these reasons, the choice of cell population to be isolated and the method of isolation used can be a significant advantage with respect to the initial purity of the mRNA isolated from the cell population.

In most tissues, glands and other organs, the cells are held together by a largely fibrous connective tissue network. This consists primarily of collagen, but may also contain - depending on the connective tissue in question - other structural proteins, polycarides and mineral deposits. When cells are to be isolated from a given tissue, it is necessary to use technical methods for releasing the cells from the "matrix" which consists of the connective tissue. Isolation and purification of specific, differentiated cell types therefore includes two main steps: Separation of cells from the connective tissue matrix and separation of cells of the desired type from all the other cell types present in the tissue.

In many cases, it turns out that the proportion of desired mRNA can be increased by taking advantage of the cells' responses to stimuli in their environment. For example, treatment with a hormone may. give rise to increased production of the desired mRNA. Other methods include cultivation at a certain specified temperature and exposure to a specific nutrient or other chemical substance. ' 2. Extraction of mRNA. An important feature of the method of the present invention is that RNase activity (ribonuclease activity) in the cell extract is substantially completely eliminated. The mRNA to be extracted is a single polynucleotide strand, which thus does not exhibit base pair binding with any complementary strand. Hydrolytic cleavage of a single phosphodiester bond in the sequence would therefore render the whole molecule unusable for transfer of an intact genetic sequence to a microorganism. As mentioned above, the enzyme RNase has high proliferation, activity and in particular stability. This enzyme is found on the skin; it survives on glassware ordinary washing operations and in some cases contaminates stores of Organic Chemicals, The problem that RNase is present as a contaminant is present in principle in all kinds of tissues, and the present procedure for eliminating RNase activity is applicable to all kinds of tissues .

According to the present invention, in combination, a chaotropic anion, a chaotropic cation and a disulfide bond disintegrant are used during the disruption of the cells and during all the process steps required to separate a substantially free protein RNA. That the agents just mentioned cooperate effectively has been shown by their use in isolating substantially undegradable mRNA from isolated pituitary cells, whereby good yields of the product have been obtained.

The choice of suitable chaotropic ions should be based on their solubility in aqueous media and on the readiness of these materials. Suitable chaotropic cations include guanidinium, carbamoylguanidinium, guanylguanidinium, lithium and the like.

Suitable chaotropic anions are i.a. iodide, perchlorate, thiocyanate, diiodosalicylate and the like. The degree of relative efficiency obtained with salts formed by combining such cat and anions depends in part on their solubility. For example, lithium diiodosalicylate is a stronger denaturant than guanidinium thiocyanate, but its solubility is only about 0.1 M and in addition this salt is quite expensive. Guanidinium thiocyanate is the preferred cation-anion combination as this salt is readily available and is highly soluble in aqueous media; the solubility is up to about 5M.

Regarding thiol compounds, such as e.g. β-mercaptoethanol, are known to break up intramolecular disulfide bonds in proteins by a thiol-disulfide exchange reaction. Many suitable thiol compounds are known to be effective for this purpose; precutonxβ-mercaptoethanol may be mentioned e.g. dithiothreitol, cysteine. propanol dimercaptan and the like. Solubility in aqueous media is necessary because the thiol compound must be present in large excess relative to the intramolecular disulfides in order for the exchange reaction to be substantially complete. Ss-mercaptoethanol is preferred, as this compound is readily available at a reasonable cost.

In the case of inhibition of RNase during the extraction of RNA from cells or tissues, the effectiveness of a given caotropic salt will be directly dependent on its concentration. The preferred concentration is therefore the highest practically possible. One reason why the present invention succeeds in maintaining mRNA intact during extraction is believed to be the rate at which the RNase is denatured, in addition to the extent to which the denaturation occurs. This should explain that e.g. Guanidinium thiocynate is a much better agent than hydrochloride, despite the fact that the hydrochloride itself is only slightly weaker as a denaturant. The effectiveness or activity of a denaturing agent is defined as the threshold concentration. required to effect complete denaturation of a protein. The denaturation rate, on the other hand, in many proteins depends on the concentration of the denaturant relative to the threshold value raised by 5th to 10th potency. See Tanford, C.A., Adv. Prot. Chem. gg, 121 (1968). qualitatively, this relationship seems to suggest that a denaturant, which has only a slightly stronger effect than guanidinium hydrochloride, can denature a protein several times faster at the same concentration. Prior to the present invention, the relationship between the kinetics of the RNase denaturation and the intrinsic retention of the mRNA molecule during its extraction from the cells should not have been discovered or exploited. If the above analysis is correct, this suggests that the preferred denaturant should be one which has a low denaturation threshold in combination with high solubility in aqueous media. For this reason, guanidinium thiocyanate is preferred over lithium diiodosalicylate despite the latter being a more potent denaturant, since the solubility of guanidinium thiocyanate is much higher and this substance can therefore be used at a concentration which allows faster RNase inactivation. The above analysis also explains why guanidinium thiocyanate is preferred over the comparably soluble hydrochloride salt: The guanidinium thiocyanate has a slightly stronger effect as a denaturant.

The use of a disulfide bond disintegrant in combination with a denaturant increases the effectiveness of the denaturant in that the RNase molecule may then be fully developed. It is believed that the thiol compound increases the course of the denaturation process in the "forward" direction by preventing rapid renaturation, which can occur when the intramolecular disulfide bonds are left intact. Ev. in addition, the contaminating RNase remaining in the mRNA preparation will remain essentially inactive, t.o.m. in the absence of the denaturant and thiol, Disulfide bond disintegrants containing thiol groups are to some extent effective at any concentration; but in general, a large excess of thiol groups relative to the intramolecular disulfide bonds is preferred in order to drive the exchange reaction toward cleavage of intramolecular disulfide bonds. On the other hand, many thiol compounds are foul-smelling and unpleasant when handled in high concentrations; there is thus a practical upper concentration limit. When using β-mercaptoethanol, concentrations in the range of 0.05M to 1.0H have been found to be effective, and 0.2M is considered optimal.

The pH of the medium during the extraction of mRNA from cells can be anywhere in the range of pH 5.0 - 8.0 - 455 794 Ä 16 After the cell degradation step, BNA is separated from the main cell-forming cell protein and DNA. Several different approaches have been developed for this purpose. All of these methods are known, and any of them is appropriate. A common prior art is a method of ethanol precipitation, in which RNA is selectively precipitated.

In the preferred technique of the present invention, the precipitation step is eliminated and the homogenate is layered directly on a 5.7M cesium chloride solution in a centrifuge tube, after which this tube is subjected. spin is described as described by Glisin, V., Crkvenjakov, R., and Byus C., Biochemistry, 2633 (1997). This method is preferred because a hostile environment is constantly maintained and REA is extracted in high yield, free of both DNA and protein.

In the processes described above, the total RNA will be purified from the cell homogenate. However, only a portion of this RNA is the desired mRNA. To further purify the desired mRNA, one takes advantage of the phenomenon that in cells of higher organisms mRNA after transcription avoids further process steps in the cell, in that polyadenylic acid is bound to this mRNA. Such mRNA, which contains bound polyn sequences thereof, can be selectively isolated by chromatography on cellulose columns with oligotymidylate bound to the cellulose - as described herein by Aviv, H. and Leder, P., loc. cit. The above procedures are most complete to yield substantially pure, intact, translatable mRNA from sources rich in RNase. Purification of mRNA and subsequent in vitro procedures can be performed in essentially one and the same manner - regardless of the type of mRNA in question and regardless of the organism that served as the source.

Under certain circumstances, for example when using tissue culture cells as a source of mRNA, contamination with R fl as ev. be insignificant enough that the method just described for RNase inhibition becomes superfluous. In such cases, ev. known methods for reducing R fl as activity should be sufficient. 3. Formation of cDNA.

The remaining steps of the process are schematically illustrated in the accompanying drawing. The first step is the formation of a_DNA sequence that is complementary to the purified mRNA. The enzyme most preferably used for this reaction is reverse transcriptase, although in principle any enzyme capable of forming a DNA strand, which is a faithful complementary strand, could be used. to the mRNA template in question. The reaction can be carried out under conditions known and described, using mRNA as a template and a mixture of four deoxynucleoside triphosphates as precursors of the DNA strand. One can conveniently ensure that one of the deoxynucleoside triphosphates is labeled with 32? in the u-position; in this way one can monitor the course of the reaction, introduce an appropriate labeling substance for the recovery of the product after such separation measures as chromatography and electrophoresis, and have the possibility of quantitative recovery for the recovery. See Ef- stratiadis, A., et al., Loc.eit.

As can be seen from the drawing figure, the product of the reaction with reverse transcriptase is a "hairpin". It has two strands, an RNA and a DNA. They are non-covalently interconnected.

The product of the reverse transcriptase reaction is removed from the reaction mixture according to known standard methods. It has been found to be expedient to use a combination of phenol extraction, chromatography on Sephadex G-10 and ethanol precipitation. "Sephadex" is a trademark (Pharmacia, Uppsala).

Once the cDNA is enzymatically synthesized, the RNA template can be removed.

There are various known methods for the selective degradation of RNA in the presence of DNA. The preferred method is alkaline hydrolysis; it is very selective and can be easily controlled by pH adjustment.

After the alkaline hydrolysis with subsequent neutralization, the 32? -Labelled eDkA may optionally, if desired, be concentrated by ethanol precipitation.

Synthesis of a two-stranded oDNA in the form of a hairpin structure is accomplished using a suitable enzyme, such as DNA polymerase or reverse transcriptase. Similar reaction conditions are used as those previously described, including the use of a u-JQP-labeled nucleoside triphosphate. Reverse transcriptase can be obtained from various sources. A suitable source is avian myeloblastosis virus. The virus can be obtained from Life Sciences Incorporated (St. Petersburg, Florida, USA), which produces the virus in agreement with the National Institutes of Health.

After the formation of the cDNA hairpin structure, it may be appropriate to purify the resulting DNA from the reaction mixture. 455 794 {s _ .. _ 18 As described above, it has been found suitable to use the steps of phenol extraction, chromatography on Sephadex G-100 and ethanol precipitation to purify the DNA product so that it is free from contaminating protein.

The hairpin structure can be converted to a conventional two-stranded DNA structure by removing the single-stranded loop that connects the ends of the complementary strands. For this purpose, there are a number of different enzymes, which are capable of specific hydrolytic cleavage of single-stranded sections in the DNA molecule. A suitable enzyme for this purpose is S1 nuclease, which has been isolated from Aspergillus oryzae. This enzyme can be purchased from Miles Research Products, Elkhart, Indiana. Here, the hànáls -DHA structure is treated with S1 nuclease, cDNA molecules are obtained in high yield, the end of which has undergone base pair formation. The extraction is followed by chromatography and ethanol precipitation as described above. The use of reverse transcriptase and S1-nuclease in the synthesis of two-stranded CDNA transcription products by mRNA has been described by Efstratiadis et al.loc. cit.

Optionally, the amount of "bluntly cut" end cDNA molecules can be maximized by treatment with E. coli DNA polymerase I in the presence of the four deoxynucleoside triphosphates. Due to the exnuclea5 and polymerase effects of enzymes, any protruding 3 'ends that may be present are removed and filled in at any protruding 5' ends that may be present. In this way, it is ensured that a maximum amount of cDkA molecules participates in the subsequent ligation reactions. In the next step of the process, the ends of the cDNA product are treated to obtain at each end the appropriate sequences containing a restriction end nuclease recognition site.

Practical details regarding the handling will be decisive for the choice of DNA fragments, which will be added at the ends. The sequence to be added at the ends is selected on the basis of the particular restriction endonuclease enzyme chosen, and this latter choice in turn depends on the choice of the DNA vector with which the cDNA molecule shall be recombined. The plasmid selected should have at least one site which is sensitive to cleavage by restriction endonuclease. For example, plasmid pMB9 contains a restriction site for the enzyme HindIII. The enzyme 455 794 19 Hind III is isolated from Haemophilus influenzae and purified according to the method of Smith, H.0. and Wilcox, K.w., J. Mol. Biol. 51, 379 (1970). The enzyme Hae III, obtained from Haemophilus aegyptuš: is purified by the method of Middleton, J.H., Edgell, M.H. and Hutcnison III, c.A., J.v1r01. io, ha (1972). that enzyme from Haemophilus suis, designated Hg; I, catalyzes the same hydrolysis specific for a recognition site. at the same recognition site as the enzyme Hind III. These two enzymes are therefore considered to be functionally interchangeable with each other.

It is convenient and convenient to use a chemically synthesized decanucleotide. consisting of two strands and containing the recognition sequence for Hind III, in order to attach this decanucleotide to the ends of the two-stranded cImA molecule.

The two-stranded decanucleotide has the sequence shown in the drawing figure. See Heyneker, H.L., et al. and Scheller, R.

H., et al. loc.cit. Those skilled in the art have access to several such sequences, which are synthetically produced and which have restriction sites, so that the ends of one of two strands of DNA can be prepared in such a way that they become sensitive to any restriction endonuclease selected. among many different such endonucleases.

The attachment of the intended sequences with restriction sites at the ends of the CDNA molecule can be accomplished by any method known to those skilled in the art. Most preferably, a reaction is used in which the connection takes place at the "dull-cut" ends (blunt needle ligation), which reaction is catalyzed by DNA ligase purified according to the method of Panet, A., et al., Biochemistry 12. , 50ü5 (1973). This type of reaction has been described by Sgaramella, V., et al-, loc.cit. The product obtained in this type of linkage reaction between, on the one hand, a cDNA with "dull cut" ends and, on the other hand, a lubricated molar excess of two-stranded decanucleotide, containing the restriction site of the Hind III endonuclease, is a cDNA, which at each end exhibits sequences with restriction sites for Hind III. When the reaction product is treated with Hind III endonuclease, a cleavage is achieved at the restriction site, whereby at the 5 'positions end portions are formed which consist of a single strand and which are self-complementary, see the drawing figure. * (455 794 20 H. Generation of a recombinant DNA transfer vector.

In principle, one could use a large number of different DNA molecules of virus type or plasmid type 1 and for the formation of recombination products with a cDNA, which has been prepared in the manner just described. The most important requirements that should be met are that the DNA transfer vector should be capable of penetrating a host cell and that it should undergo replication, and that it should have such a genetic determinant as to enable the selection of the host cells which have received the vector. However, for general safety reasons, the selection should be limited to those transfer vectors which are considered suitable for the type of experiment in question, in accordance with the above-mentioned ^ NIH regulations. The list of approved DNA transfer vectors is constantly being expanded as new vectors are developed and approved by the NIH Reoombinant DNA Safety Committee.

Within the scope of the present invention is the use of any viral DNA and plasmid DNA which have the properties described above, including those which may only later be approved by the NIH. Suitable transfer vectors, currently approved for use, include various derivatives of bacteriophage lambda (see, e.g., Blattner, FR, Williams, BG, Blechl, AE, Denniston-Thompson, K., Faber, HE, Furlong , LA, Grundwald, D.J., Kiefer, D.0., Moore, DD, Schumm, Jw, Sheldon, EL and Smithies, O., Science 196, 161 (1977) and derivatives of the plasmid col El, see t. eg nøariš fl gz, nL, Bolivar. s., Gcoaman, nn, aoyer, nw and Betlach, MN, ICN-UCLA Symposium on Molecular Mechanisms In The Control of Gene Expression, DP Nierlich, WJ Rutter, CF

Fox, Eds. (Academic Press, NY, 1976), p. # 71-H77. For plasmids derived from col E1, it is characteristic that they are relatively small, have molecular weights of the order of a few million and have the property that the number of copies of plasmid DNA per host cell can be increased from 20-H0 under normal conditions to 1000 or more than 1000 by treating the host cells with chloramphenicol. Because the gene dose in the host cell can be increased, it becomes possible under certain circumstances and under the control of the researcher to get the host cell to primarily produce such proteins, the code of which is provided by genes in the plasmid. Such derivatives of col E1 are therefore used in the tubing of the present invention. Suitable derivatives of col E1 include the plasmids pNB-9, which carry the gene for tetracycline resistance, 455 794 21 and pBR-513, pBR-315, pBR-316, pBR-317 and pBR-322, which in addition to the gene for tetracycline resistance contains a gene for ampieillin resistance. By the presence of the drug-conferring genes in question, it becomes possible to easily and appropriately select cells which have been successfully infected with the plasmid, since colonies of such cells grow in the presence of the drug. whereas, on the other hand, such cells which have not received the plasmid will not grow or form colonies. In the experiments reported in the present specification as specific examples of the present invention, a plasmid derived from col E1 was always used, which in addition to the "label" described above with respect to drug resistance contained a site for Hind III.

Regarding the host organism, exactly as in the case of plasmids. that there are a large number of organisms that could in principle be selected, but with regard to public safety, the choice is very limited. A strain of E. coli, designated X-1776, has been developed and approved by the NIH for use in procedures of the type contemplated herein, in accordance with the safety regulations of P2. See Curtiss. III, R., Ann.Rev.M1crobiol .. 30, 507 (1976). E. coli RR-1 is suitable when the safety regulations according to P3 can be followed. In the same way as in the case of plasmids, with regard to host cell strains, it is possible according to the invention to use any such strains which can serve as recipients for the selected vector.

Recombinant plasmids are formed by mixing plasmid DNA treated with restriction endonuclease with cDNA containing similarly treated end groups. In order to eliminate the risk to the greatest possible extent. that segments of cDNA form combinations with each other, plasmid DNA is added in molar excess relative to eDNA. In prior art methods, this has resulted in most plasmids undergoing cyclization without the incorporation of any eDNA fragment. The cells subsequently transformed contained mainly plasmid, and non-cDNA recombinant plasmids. The selection process was therefore a very cumbersome and time consuming procedure. In the prior art methods, attempts have been made to solve this problem by trying to obtain DHA vectors with a site of restriction endonuclease in the middle of a suitable labeling gene, so that the introduction of a recombinant divides the gene, thereby the function for which the gene is a code is lost.

Preferably, a method is used to reduce the number of colonies, from which suitable recombinant plasmids are to be selected. According to this method, restricted endonuclease treated plasmid DNA is treated with alkaline phosphatase, an enzyme which is on the market and can be obtained from various manufacturers, e.g. Worthington Biochemieal Corporation, Freehold, New Jersey. USA. Treatment with alkaline phosphatase removes the phosphates present at the 5 'end positions from the ends of the plasmid formed by the middle zendonuclease and prevents self-priming of the plasmid DNA in question. Ring formation, and thus conversion, will thus become dependent on the insertion of a DNA fragment, which contains 5 'phosphorylated ends. The described procedure reduces the relative conversion rate at non-recombination to less than l: 10 + u.

The present invention is based on the fact that the DNA ligase catalyzed reaction occurs between a 5 'phosphate end group of a DNA molecule and a jihydroxyl end group of a DNA molecule. If the phosphate group in the 52 position is removed, no bonding reaction takes place. With regard to the binding of DNA with two strands, three situations are possible, as shown in Table 1 below. 23 {455 794 'lelzelLl Cases Reaction components Ligase product I BI 5! 3! 5 '--- ~ OH Hq0} P0 ------ --- 0-PO - (_ ---_ OPOBHQ + HO_____il _ O_P_O_ + 2H20 5' 3 '5' 5 'II 3! 5! 3 ! 5; ___. On no Po ._. --- _-- ----. OPo ~ «--_ 0H + 2 30H - ~~ --- __--- OH HO -« ---- ¿ H2O 51 3 / s! J! III j '5' '____ “OH H0 no reaction on“ ”1110-- 5' 3 'Table 1 shows DNA, which has two strands, schematically by means of a pair of parallel solid lines. The 5 'and 3' end position groups are shown to carry either a hydroxyl group (OH) or, where applicable, a phosphate group (OPO3H2).

In case I, 5 'phosphates are present at both reacting ends, with the result that both strands are covalently bonded together. In case II, only one of the strands has a 15 'end position - an existing phosphate group, which results in a single strand being covalently bound, a gap appearing at the same time in the other strand. The non-covalently linked strand is held together with the linked molecule by hydrogen bonds between complementary base pairs on the opposite strands, as is generally known. In case III, there is no 5 'phosphate group at either end of the other component, with the consequence that no reaction can occur. Therefore, undesired bonding reactions can be prevented by treating the reactive ends in question, the bonding of which it is desired to prevent, and for removing the 5 'phosphate groups from these ends. For this purpose, any method can be employed which is suitable for the removal of 5'-phosphate groups and does not otherwise damage the DNA structure. Hydrolysis, which is catalyzed by the enzyme alkaline phosphatase, is preferred.

The method just described is also suitable if a linear DNA molecule is to be cleaved to form two sub-fragments - typically using a restriction endonuclease enzyme - and then reconstituted with the original sequence. The sub-fragments can be purified separately, and the desired sequence can be reconstituted by reconnecting the sub-fragments.

The enzyme DNA ligase, which catalyzes the end-to-end connection of DNA fragments, can be used for this purpose. See Sgaramella, V., 'Van de Sande, J.H. and Khorana, H.G., _ Proc.Natl.Acad.Sci.USA §Z¿ 1468 (1970). If the sequences to be linked do not have "bluntly cut" ends, the ligase obtained from E. coli can be used, see Modrích, P. and Lehman, I.R., J. Biol. Chem. šï§¿ 5626 (1970).

The efficiency with which the original sequence is reconstituted based on sub-fragments obtained by treatment with restriction endonuclease. is significantly increased if one uses a method for preventing reconstitution with incorrect sequences. Said undesirable results are avoided by the cDNA fragment. which has the desired sequence and which is homogeneous in length, is treated with a means for removing the phosphate groups present at the 5 'end position on the cDNA, before this homogeneous cDNA is cleaved with a restriction endonuclease.

The enzyme alkaline phosphatase is preferred. The phosphate groups existing in the 15 'end positions are structural conditions for the subsequent binding action exerted by the enzyme DNA ligase, which is used in 1, and for the reconstitution of the cleaved sub-fragments. Thus, ends without 5 'phosphate groups cannot be covalently bonded together. The DNA sub - fragments can only be linked together at ends, containing a 5 'phosphate, which is formed by the cleavage performed on the isolated DNA fragments by restriction endonuclease. The above method prevents the most important of the undesirable bonding reactions from being avoided, namely that the two fragments are bonded in reverse order, i.e. from behind to forward i.st.f. from side to back. Other side reactions that may occur, such as dimer formation and cyclization, are not prevented: they can occur through a type II reaction, see Table 1 above. However, the inconvenience of these side reactions is not as great, since these reactions lead to physically separable and identifiable products, whereas recombination in reverse order does not lead to such products.

Example Gel 1. (Reference Example) Here, isolation and purification of a DNA is described. which has the entire structural gene sequence for RGH, as well as the synthesis of a transfer vector. which contains the entire structural gene for RGH, as well as the construction of a microorganism strain. which contains the gene for RGB as part of its genetic equipment.

In the case of genes of non-human origin, the US federal authorities do not require that cDNA be isolated in such high purity. which is required 1 Rehearse human cDNA. Therefore, it was possible to isolate the cDNA. which contains the entire RGH structure gene. by isolating electrophoretically separating DNA »down the vesntafae length. ca 8oo baspar. determined on the basis of the known amino acid length of the RGH molecule. As a source. for RGH mRNA, cultured rat pituitary cells were used. a suclone to the GH-1 cell line, available from the American Type Culture Collection. See Tashjian, A.H., et al., Endocrinolocv§§¿ BÄE (1968). When such cells are grown under normal conditions, growth hormone mRNA constitutes only a small percentage, 1-3%, of the total poly-A-containing RNA. However, the level of growth hormone mRNA was increased to a value higher than that of other cellular mRNAs through synergistic interaction of thyroid hormones and glucocorticoids. RNA was recovered from 5 μg of 10 cells grown in suspension culture and brought to produce growth hormone by incorporating 1 mM dexamethasone and 10 mH L-triiodothyronine in the culture medium for 1 day before cell recovery. Polyadenylated RNQ was isolated from the cytoplasmic membrane fraction of the cultured cells, as described in the literature. 455 794 26 See Martial, J.A., Baxter, J.D., Goodman, H.H. and Seeburg, P.H., Proc.Nat.Acad. Sci. USA Z 18¿ 1816 (1977), and Bancroft, F.C., Wu, G. and Zubay, G., Pr0c.Nat.Acad.Sci. USA: go} 6U6 (1973).

The resulting mRNA was further purified and transcribed into two-stranded cDHA. After fractionation by gel electrophoresis, a weak but clear band could be observed. corresponding to a DNA that had a length of about 800 base pairs.

When the total cDNA. obtained by transcription from the cultured pituitary cell mRNA. treated with Hha endonuclease, two larger or major DNA fragments were obtained when the product was subjected to electrophoretic separation. One fragment (fragment A) was about 320 nucleotides in length, and the other fragment (fragment_B) was about 2H0 nucleotides in length. When both fragments A and B were subjected to nucleotide sequence analysis, it was found that these fragments did indeed form part of what must be considered the RGB coding region based on published data regarding the amino acid sequence in RGH and in comparison with other known growth hormone amino acid sequences. See Wallis, N. and Davies, R.

V.N., Growth Hormone And Related Peptides (Eds., Copecile, A. and Muller, E.E.), p. 1-lä (Elsevier, New York, 1976), and Dayhoff, M.0., Atlas of Protein Sequence and Structure, §¿ suppl. 2, pp. 120-112 (National Biomedical Research Foundation: Washington, D.C., 1976). When the cDNA electrophoretically isolated in the manner described above - which consisted of two strands and had a length of 800 base pairs - was similarly subjected to treatment with Hhal- endonuclease, two fragments were found among the main products from the cleavage, which in terms of their length corresponded to fragments A and B.

Because the approximately 800 base pairs containing RGB-CDHA were not purified using restriction endonuclease. was it necessary to subject this DNA to a treatment to remove any molecular ends with a single unpaired strand. In practice, the treatment was performed to remove such unpaired ends before electrophoresis separation. 1 Päßul 60 mh Tris-HCl, pH 7.5, 8 mH MgCl 2, 10 mH ß-mercaptoethanol, 1 mM ATP and 200, uH of each of the phosphates dATP, dTTP, dGTP and dCTP. The mixture was incubated with 1 unit of polymerase I for DNA from E. coli at 10 ° C for 10 minutes to exonucleolytically remove any existing ends projecting in the 3 'position and to achieve a "filling" at all possible protruding ends in the 5 'position. DNA polymerase I can be purchased from Boehringer-Mannheim, Biochemicals, Indianapolis, Indiana, USA.

The approximately 800 base pairs containing RGH cDNA were treated by the addition of chemically synthesized Hind III links. Plasmid pBR-322, which has a gene for ampicillin resistance and has a single Hind III site, which is within the tetracycline resistance gene, was pretreated with Hind III endonuclease and alkaline phosphatase.

The treated plasmid was reacted with the 800 base pair containing RGH cDNA in a DNA ligase reaction mixture. The ligase reaction mixture was used to convert a suspension of E. coli cells X-1776. Recombinant colonies were selected by culturing on plates, containing nutrients and ampicillin, and on the grounds that the cells could not grow at 20 pg / ml tetracycline.

10 colonies of this kind were obtained; all of these contained the plasmid with a section of about 800 base pairs inserted therein, which was released by Hind III cleavage.

The 800 base pair containing RGH DNA was isolated in preparative amounts from the recombinant clone pRGH-1, and its nucleotide sequence was determined. This time, the nucleotide sequence included portions of the 5 'untranslated region of RGH as well as a sequence of 26 amino acids, which is present in the growth hormone precursor protein prior to secretion. With the exception of positions 1 and 8, the predicted amino acid sequence shows good agreement with the partial amino acid sequence of rat growth hormone, as described by Wallis and Davies loc. cit. This partial sequence comprises residues 1-43, 65-69, 108-113, 133-143 and 150-190. 455 794 28 Exemgel 2.

This example describes the isolation and purification of the entire gene sequence that codes for HGH (human growth hormone), as well as the synthesis of a recombinant plasmid containing the entire structural gene for HGH, and the production of a microorganism which, as part of its genetic equipment, contains the entire structural gene for HGH.

The isolation of HGH mRNA is mainly carried out in the manner described in Example 1, however in the present case the biological source material is human pituitary tumor tissue. Five benign human pituitary tumors, which are rapidly frozen in liquid nitrogen after removal during surgery. weighing each 0, N g - 1.5 5, thawed and homogenized in HH guanidinium thiocyanate, containing 1M mercaptoethanol and buffered to pH 5.0, at HOC. The homogenate was layered over 1.2 ml of 5.7 M CsCl, containing 100 mM EDTA, and centrifuged for 18 hours at a speed of 37,000 rpm in the SW 50.1 rotor of a Beckman ultracentrifuge at 1500 (Beckman instrument Company, Fullerton, California, USA). RNA moved toward the bottom of the tube. Further use of a column of oligo-dT and sucrose gradient sedimentation was performed.

About 10% of the RNA thus isolated was thought to give a code for growth hormone - this on the basis of a sample, where a radioactive amino acid precursor was incorporated into a precipitated anti-growth hormone material in a cell-free translation system obtained from wheat germ. See Roberts, B.E. and Patterson, B.M., Proc.Hat.

Acad. Sci, USA. Zgè 2330 (197)). Preparation of HGH-c fi NA is mainly carried out in the manner described in Example 7. The resulting HOB cDNA is fractionated by gel electrophoresis, and materials migrating to a position corresponding to a length of about 800 nucleotides are selected for cloning. The selected fraction is treated with DNA polymerase I ,. after which it is subjected to the addition of Hind III links in the end stands. This cDNA is then recombined with plasmid pßh-522, which has been treated with alkaline phosphatase. The recombination takes place with the help of DNA ligase. With the resulting recombinant DNA, E. coli X-1776 is converted, and a strain containing HGH-DHA is selected. This strain is grown in preparative amounts, HGH DNA is isolated from the strain and its nucleotide sequence is determined. The cloned HGH DNA is found to comprise nucleotides, which encode the entire amino acid sequence of HGH. The 4sse794 "29 first 23 amino acids 1 HGH are HqN-Phe-Pro-Thr-Ile-Pro-Leu-Ser-10 -Arg-Leu-Phe-Asp ~ Asn-Ala-Met-Leu-Arg-Ala-His -Arg-Leu-His-Gln-Leuf.

The rest of the sequence is shown in Table 2.

Table 2.

The nucleotide sequence of an HGH DNA strand. The numbers refer to the amino acid sequence of HGH starting at the amino terminus. The DNA sequence shown corresponds to the mRNA sequence of HGH, with the exception that an mnNA 1 instead of T contains u. '.N «|||| uuuoæuukoæuCuPu <@ Uuu @ uuvu »za> Hu mßu“ um »Hu: Ho fl ~> fi m fi Hua uuu uuk: nom wu <nau: au ~ c> u fl u wu <: ua man use: Mu fl m> maa nn <uu: nm <maa w »<usa nav uah: ua: ua æ fl o nav: m <maa om fl. ow fi. _, ueu <»u: mg: ud - <@m <maa =« <w «=» um = «<» za @w <»pm Mag uuw ~>« ~ = a_ = Hu mä; »Gm» HH: Hu »Hu hav» kw on »» um »Hu. ce "u nm <s fl o: ua wu <aan uu: som una: # 0 u fl u a fl u s fi u: ao: ua nm <m> A: wa: uu mm <une ~ m>: m <now nm $ .num w ~ <> ~ u o-. uu> P.H fl>: uQ. &% W.:m <dn <Mon An> nam wu <: un msn: HU Hn> Ohm: ao: uu unk hum: ab uHH: ua : mä: UA hum UHH mh <cø fl _ Oæ _ uhu uno o: un: uu: uu: ua: m <nam maa: Hu: Ho uzß: Hc: Hu wu <cm <»um oum use cum man nam: Ho now osm mao: uu Ham usa 1 ow o: Hu nu »= w <= ~ u: uu u = ~“ um »ÄH mä; = ~ u: au mäd eu» QHH u> @ «A <: Hu: Hu »Gm: Hu: Hu p> @» ne @m <»za« H <. _ N «° <.« N «~ _ _ _ 455 794

Claims (4)

'455 794 31 P a t e n t k r a v Plasmid suitable for transfer of a gene to an Escherichia coli bacterium, characterized in that it contains in its nucleotide sequence a sequence encoding the amino acid sequence of human growth hormone and comprises 5 '--- TTR, CCLQACLSATM4CCLSXGTYGQ9, s5 , 0cAx1, AAx12 GCL1sÅTG14X1STY1sW1GGZ1eGCL1vCAK1sW19Gz: 9X2oTY2oCÅK21CAJ22X2s TY23GCL24TTK25GÅK25ÅCL27TÅK23CAJ29GAJ39TTK33GAJ32GAJ33GCL34 TÅK35ÅTM35CCL37AAJ33GÅJ39CAJ40ÅAJ41TAK42QR43S43TTK44X45TY45 CÅJ4sAÅK47CCL4sCAJ49ACLsoQRs1Ss1Xs2TYs2TGKssTTKs4QRss5ssGAJse QRs1551ATMssCCLssÅCLsoCCLs1QRs25e2AAKssWe CAJsaCÅJssAA31oQR11571AAK12X7aTY1sGA314XvsTY7sX7eTY1eW1vGZ11 ÅTM1sQR19519XsoTYsoXs1TYs1Xs2TYs2ÅTMsaCAJs4QRss5ssTGGseXa1 TY57GAJ83CCL59GTL90CÅJ91TTK92X93TY93W94GZ94QR95S95GTL95TTKg7 GCLesAÅK99 ^^ K10g X1o1TY1o1GTL1o2TAK1oaGGL1o4GCL1osQR1os51os GO: o1QR1os51osAÅK1oeGTL11oTÅK1: 1GAK112X: 1aTY11sX: 14TY114 AAJ11sGÅK11eX1: 7TY111GÅJ11sGAJ11eGGL12oÅTM121CAJ122ACL12s X124TY124ATG12sGGL12sW121GZ127X12sTY12sGÅJ: 2sGÅK1soGGL1s1 QR13 25: 32CCL1s3W134GZ1s4ACL1ssGGL: 36CAJ: a7ATM1ssTTK13sAÅJ14o CÅJ141ACL142TÅKJ4sQR1445144AAJ14sTTK14eGAK147ACL14sAAK14s QR1so31soCÅK1s: AÅK1s2ÛÅK1ssGÅK1s4GCL1ssX1ssTY1ssX1s7TY1sv AAJ1ssAAK1ssTAK1eoGGL1s1Xie2TY1s2X1esTY1eaTAKzs4TGK: ssTTK1se W1e1GZ1s1AAJ1esGAK1esATG17oGÅK1v1AAJ172GTL11aGAJ: 14ACL17s TTR11eX: 11TY111W11aGZ11sÄTN11sGTL1soCÅJ1s1TGK1szW1ssGZ1sa QR1s451s4GTL1ssGAJssGGL1a7QR: SAS: ssTGK1ssGGL1soTTR¿f * 3 'where is deoxyadenyl, is deoxyguanyl, is deoxycytosyl, is thymidyl, A or G; is T or C; is A, T, C or G; is A, C or T; XZHNCJV-BOOIP 'is T or C' ° m Yn is A or G, and C if Y is C I! IJ '455 794 32 or T; Yn is A, G, C or T if Xn is C, and A or G if Xn is T; Wn is C or A, if Zn is G or A, and C if Z "is C or T; Zn is A, G, C or T, if Wn is C, and A or G if Wn is A; QRn is TC , if Sn is A, G, C or T, and AG if Sn is T or C; Sn is A, G, C or T, if QRn is TC, and T or C if QRn is AG and the index numbers, n, indicates the position of the amino acids in human growth hormone, for which the nucleotide sequence is the equivalent in accordance with the genetic code, the amino acid positions being numbered based on the amino terminus. Escherichia coli bacterium modified to contain the plasmid according to claim 1. A method for producing a plasmid according to claim 1, characterized in that (a) mRNA is isolated from pituitary cells by homogenizing the pituitary cells in the presence of an RNase inhibitor composition containing 4M guanidinium thiocyanate and 0.0S ~ 1.0M 8-mercaptoethanol at pH 5.0-8.0L thereby preventing RNase degradation of this mRNA; (b) producing cDNA with a nucleotide sequence encoding growth hormone by treating recovered mRNA with a reverse transcriptase in a manner known per se, after which this CDNA is double-stranded; (c) attaching sequences with a recognition site for a restriction endonuclease to the ends of the resulting double-stranded CDNA, the restriction endonuclease being the same as in step (d) below, after which the resulting CDNA is cleaved with the restriction endonuclease to form cohesive ends; (d) a bacterial plasmid is opened with restriction endonuclease, eg Hind III or Hsu 1, to form an opened plasmid vector with cohesive ends; (e) The 5 'phosphate end groups of the plasmid vector obtained in step (d) are hydrolyzed by treatment with alkaline phosphatase, for example at pH 8.0 and the temperature 65 ° C for 30 minutes, whereby those under (d ) said cohesive ends are rendered incapable of bonding to each other; and (f) the plasmid vector treated in step (e) is linked to the cDNA obtained in step (c) by incubation in the presence of DNA ligase and ATP, for example at pH 7.6 and the temperature 14 ° C for 1 hour with use of a molar excess of the plasmid vector, thereby obtaining a plasmid having a nucleotide sequence encoding growth hormone. A method of producing an Escherichia coli bacterium containing a plasmid having a nucleotide sequence encoding human growth hormone, characterized in that the bacterium is combined with a plasmid according to claim 1.