DD154023A5 - METHOD FOR PRODUCING A DNA TRANSFER VECTOR - Google Patents

Abstract

The present invention relates to the isolation of a specific nucleotide sequence containing the genetic code for a specific protein, the synthesis of DNA with this specific nucleotide sequence and the transfer of this DNA in a host microorganism in which replication of the DNA occurs. In particular, the invention relates to the isolation of growth hormone genes, their purification, transfer and replication in a microbial host and their subsequent characterization. The invention provides new products. These include a plasmid into which a specific nucleotide sequence derived from a higher organism is inserted, and a new microorganism which, as part of its genetic makeup, contains a specific nucleotide sequence from a higher organism.

Description

The present invention "relates to the preparation of a DNA transfer vector with a nucleotide sequence encoding growth hormone",

Characteristic of the known technical solutions

The following symbols and abbreviations are used in this description %

DN-S ~ deoxyribonucleic acid

RNA = 3 ribonucleic acid.

cDNA = complementary ONS

(enzymatically synthesized from a mRNA sequence)

'KiRNS =' messenger RNA

tRNA - Transfer RNA "

dATP w doxyadenosine triphosphate

dGTP ™ deoxyguanosine triphosphate

dCTP = deoxycytin trisphosphate

A ~ Adenine

T ~ Thyrain

G κ guanine

C a 'Cytosine'. ,

T r 1 s - 2 era i si ο - 2, - h yd r ο χ y »ethy I ·» 1. Z4 - ρ r ο ρ a η di ο 1

EDTA ~ ethylenediaminetetraacetic acid.

ATP S = adenosine triphosohet

TTP - thymidine triphosphate

The biological significance of the base sequens; aar DNS is the

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a Memory of Genetic Information "It is known that the base sequence of the DNA is used as the code that determines the amino acid sequence of all the proteins produced by the cell. In addition, portions of the sequence serve regulatory purposes to control the timing and amount of each protein Finally, the base sequence in each "S-strand serves as a template, the replication of the DNA that accompanies cell division."

The way in which the information of the Sasensequenz-DNA is transferred to the amino acid sequence of proteins is a fundamental process that universally for all living organisms "

It has been demonstrated that any amino acid commonly present in proteins is determined by the sequence of one or more "trinucleotides or triplets." The protein thus corresponds to a segment of the DNA with a triplet "sequence corresponding to the amino acid sequence of the protein", The genetic code is illustrated in the following table %

Genetic code

Phenylalanine (Phe) TTK Histidine (His) CAK

Leucine (Leu) XTY Glutamine (a) CAD

Isoleucine (lie) ATM asparagine (Asri) AAK

Methionine (Met) ATG lysine (Lys). AAO

Valine (VaI) '' GTL aspartic acid (ASp) GAK

Serine (Ser) ORS Glutamic Acid (Glu) CAD

Proline (Pro) CCL Cysteine (Cys) TGK

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Threonine (Thr) ACL Tryptophan (Try) TGG

Alanine (Ala) GCL Arginine (Arg) VVGZ '

Tyrosine (Tyr) TAK Glycine (Gly) GGL

Termination signal TAD.

Termination signal TGA

Key's Oedes triplet of three letters represents a trinucleotide of DNA, with a 5'-end on the left and a 3'-end on the right «» The letters stand for the purine or pyrimidine bases, which form the nucleotide sequence σ

A - adenine

G = guanine

, C = cytosine

T = thymine

X = T or C if Y = A or G

X a'C if Y = C or T

Y s A, G _f C or T, if X = C 'Υ »A or G, if X« T

WaC or A if Z = A or G

W-C, if ZsC or T

Z = A, G ₁ C or T if W = C

Z a A or G ₁ if W = A QR a TC _f if S ~ A, G, C or T QR a AG, if ST or C

S ~ A ₁ -G, C or T if QR »TC

S-β τ or C, if QR = AG

D ~ A or G

K s T or -C

L ~. A _s T _e C or G

M - A, C or T.

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In the biological process of the upper setting of the nucleotide sequence into an amino acid sequence, the so-called Transcription is carried out as the first stage _ö In this step, a segment of DNA with the competent for the protein to be prepared sequence transferred to an RNA, the RNA is a DWS ahn- pending polynucleotide in However, ribose instead of deoxyribose and uracil instead of thymine 8teh © n _e The bases of the RNA are capable of the same type of base pairing as the bases of the DNA. The RNA resulting from transcription of a DNA nucleotide sequence is therefore complementary to this copied sequence RNA is called messenger * "RNS (MRM) because of its intermediate position between dsm genetic apparatus and the apparatus of the protein synthesis of the cell"

In the cell, the mRNA serves as a template in a complex process involving OEEM numerous enzymes and organelles within the Zeil © and specific for the synthesis of amino acid sequences results, "This process is known as translation

Often there are further steps which serve to _{t f} of aer resulting in translation amino acid sequence to produce a functional protein tional = "An example is the production of insulin". -.

The direct precursor of insulin is a polypeptide _for the probe containing the two insulin chains A and B bound via another peptide C, see Steiner, D _e Fe-. Cunningham, D _if Spigelman, L _s and Αΐβη, - Β ** Science 3.57, 597 (iQ67) _# Recently it was reported that the first trans-

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For example, the product of ligation of xnsulin mRNA is not proinsulin itself, but is a pre-proinsulin containing more than 20 other amino acids at the amino terminus of proinsulin, compare Yahn, S * D _{e (} Germ, P ₆ and Steiner, D ₀ F _c , Proc * Natl · Aead «See» USA 73, 1964 (3.976), and Lomedico, P _e T _e and Saunders, Ge F * _f Nucl «Acids Res * 3, 381 (197δ) _β The structure of the pre-proinsulin molecule can schematically represented as follows : V'ierden * NH ₂ - (Pre-peptide) -B chain ~ (C-peptide) -A-chain © -> COOH _e

Numerous proteins important for medicine ODSR research are found in the cells of higher organisms such as vertebrates or produced © These include, for example, the hormone insulin, other Peptidhonnone as growth hormone involved in the regulation of blood pressure proteins and numerous enzymes with important technical, Medical or Research _Purposes It is often difficult to obtain these proteins in appropriate amounts by extraction from the organism, especially proteins of human origin. There is therefore a need for techniques whereby such proteins can be released from cells outside the organism reasonable quantities are generated * In some cases it is possible _£ to obtain suitable cell strains through the techniques of tissue culture, "However, the growth of cells in.Gewebckultursn is slow, the medium is expensive" conditions must be strictly controlled, and the exploiters ; are low "Often it is also difficult to obtain a cultured cell strain with the desired differentiated properties *

In contrast, microorganisms such as bacteria are relatively easily bred in chemically defined media. "

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* The fermentation technology is already highly developed and well manageable. The growth of organisms is rapid and high yields are possible. In addition, certain microorganisms have already been genetically well characterized and indeed belong to the most characterized and recognized organisms.

Reverse transorptase catalyzes the synthesis of RNA template-complementary DNA in the presence of the RNA template, © in the Qligo-deoxynucleotide protiroer and the four deoxynucleic acid triphosphates dATP., DGTP "dCtP and TTP. The reaction is initiated by the non-RNA template. covalent binding of the oligo-deoxynucleotide Ppioiers to the 3 'end dor mRNA, this is followed by the gradual addition of the appropriate Doxynucleotide ent, "speaking of the base-pairing rule, to the 3' end of the growing chain" the resulting molecule as a product can be used as hairpin The reverse transcriptase may further catalyze a similar reaction in which a DNA-Eirizel strand serves as a template, in which case the product is a hairpin-shaped DNA double strand obtained with a Loop DNS single strand through which a pair of ends are joined; compare Aviv, H _f and Leather, P ₆ , Proc «Nati» Acad. Let us say: * USA 69, 1408 (1972) and Efstratiadis, A., Kafatos, F * C ₅₅ Maxam, A «F ₀ . and Mania · tis, T _e Cell, 7, 279 (1976).

Restriction endonucleases are enzymes capable of hydrolyzing phosphodiester bonds in double-stranded DNA, cleaving the sequence of the DNA strand. * If the DNA is in the form of a closed loop, so

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this is converted into a linear form. "The main feature of an enzyme of this type is that the hydrolytic effect occurs only where a specific nucleotide sequence exists." This sequence is called the recognition site (cleavage) of the restriction endonuclease. "Restriction endonucleases Several restriction endonucleases hydrolyze the phosphodiester bonds in both strands in the same position so that blunt bonds _t are a few nucleotides apart, creating single-stranded regions at each end of the cleaved molecule These single-stranded ends are self-complementary, therefore, cohesive, and can be used to re-bind the hydrolysis-related DNA. Since any DNA susceptible to cleavage by such an enzyme must contain the same recognition site same n cohesive ends produ ~ ed s so that it is possible - straight logo DNA sequences with others, to combine sequences similar to those obtained; see Roberts, D. R * _Sf * Cr.it Rev _e Biochenu 4 "123 (1976)," The recognition sites are relatively rare, however the general utility of the Restrictions endonucleases has been greatly enhanced by the chemical synthesis of oligo-nucleotides doppelsträngi- ger with the sequences of Erksnnungsstel "len * Thus, virtually any ONS Segraent be linked to another segment _t by simply match the ·" de Restrictions-Oligu-Nuclöotid to Diss molecule ends appends .and the product to the hydrolytic action 'dor corresponding Restrictiono -Endonuclease, yielding the necessary cohesive ends; compare Heynecker, Shine j _Η β L _e, Goodman, CJ, "Boyer, Η" Μ _{"e Rosenberg's} li® W _0, De ₁ Dickereon, - Π" · £ _'s Narang _£ S _ffi A *, rtakura, K ₀ , Lin * S ₄

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and Riggs * A ₉ D *, Nature 263 _f 748 (1976), and Scheller. R. Κ _{β #} Oicksrson, R * Ε «', Boyer« H ₀ W ₆ , Riggs. A. D _e and • Itakura, K _0f Science'196, -177 (1977).

Sl-endonuclease is an enzyme capable of hydrolyzing the phosphodiester bonds of single-stranded DNA or single-stranded intermediates in otherwise double-stranded DNA; compare Vogt, V _c , M *, £ ur _c 3 «Biochenu 33, 192 (1973) ·

DNA ligase is an enzyme that causes the formation of a phosphodiester bond between two DNA segments with a 5'-phosphate or a 3'-hydroxyl, for example, formed by two ONS fragments held together by cohesive ends be "normal function of the enzyme is presumably to join single-stranded intermediate pieces in an otherwise double-stranded DNA molecule" Under appropriate conditions, however, DNA ligase, the compound blunt ends catalyze _f where two molecules are bound covalently with blunt ends; compare Sgaramella, V _e , Van de Sande, 0 " H ₀ and Khorana, H _e 6", Proc. Natl * Acad ₆ Sei, USA 67, 1468 (1970) «

Alkaline phosphatase is an enzyme which _t Phosphatesterbindungen _{including the 5'-terminal phosphate groups in DNA hydrolyzed "

Another step in the overall process to be described is the insertion of a specific DNA fragment into a DNA vector, for example a plasmid. "A plasmid is any autonomously DNA" replicating entity that can be present in a microbial cell adjacent to the genome of the host cell. A plasmid is not genetically linked to the host cell chromosomes _f plasmid deoxyribonucleic acid

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acids are double-stranded ring molecules _f. generally with molecular weights of the order of a few million, although in some the molecular weight is greater than 10. They usually represent only a small percentage of the total DNA of the cell. The piasmide DNA can be resolved * separate generally & from the DNA of the host cell due to the large large enunterschiedes the plasmids can regardless of the speed aer division replicate aer host cell, and in some cases their replication rate can be influenced by the experimenter by altering the growth conditions. Although the plasmid is a closed ring, one can artificially introduce a DNA segment into the plasmid to produce a recombinant plasmid of increased molecular size without substantially impairing its ability to replicate or express any genes of the plasride. The plasmid therefore serves as a useful vector for transferring a DNA secretion into a new host cell. Plasmids suitable for recombination of DNA typically contain genes which may be useful for selection purposes, for example, genes of the drug resistenζ

To Illus'trierung practicing the invention, the isolation and the transfer of rat insulin gene are described in detail * -The .Insulin, was chosen in this case because of its central importance of d r point of view of clinical medicine and from the perspective of 'basic research' The disclosed method may be readily understood by one of ordinary skill in the art of isolating insulin gene from other organs, including humans. transmitted

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- 10 genes will be «

Insulin was first isolated in 1922. "It is well known that this hormone is used today to treat diabetes. Although the slaughterhouses deliver pancreas from cattle and pigs as insulin sources, there is a shortage of the hormone as the number of diabetics increases." develop some diabetics to produce comprehensive sets an allergic reaction to beef and Schweineinsuiin "· the possibility of human insulin in the .weltweiten demand, would be highly desirable * Dio producing human insulin in bacteria is a technique that achieves this desired goal could be "a progress in this direction has ever been" but previously thwarted that no technique for the introduction of the insulin gene in B kterien _a existed "the present invention provides such a technique provides"

The possibility of producing a DNA having a specific sequence den'genetischen code for a specific protein emits the modification of the nucleo-'tidsequenz by chemical or biological means permits such Aab and the protein produced is finally modified "In this way, could For example, to make a modified insulin that caters to specific medical needs "The genetic ability to produce an insulin-like amino acid sequence with the essential functional properties of insulin can therefore be transferred to a microorganism." Similar considerations apply in the case of growth hormones.

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The ability to transfer the genetic code for a specific protein that is needed in the normal metabolism of a higher organism into a microorganism such as a ßakte-Ministry opened: new possibilities for the production of such proteins ,, This creates the possibility for propagation or to the replacement of such proteins by proteins produced by microorganisms modified according to the invention where the ability of the higher organism to produce normal production of these proteins has been impaired, and the possibility arises, for example, of synthesizing symbioses between the organisms of the invention _f compensate microorganisms and Mens Chan with chronic or acute deficiency diseases., the inventive manner of genetically modified microorganisms implanted or otherwise incorporated to the pathological metabolic error "

It would therefore be highly desirable to obtain the transfer of gene code for a protein of medical importance from an organism which normally produces the protein into a suitable microorganism; in this way the protein could be produced under controlled growth conditions by the microorganism, and in obtain desired quantities wordene It is also jnöglich that the total cost of producing the desired P _r oteina could be considerably reduced by such a method * the ability to isolate and transfer aor gene sequence, which determines the production of a particular protein in a Microorganism of known genetic structure.

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net also valuable opportunity to examine the research, how to control the synthesis of such a protein and how the protein is processed after synthesis. Furthermore, isolated genes can be altered to encode protein variants with other therapeutic or functional properties. ,

The present invention relates to measures to achieve the stated objects. A process is disclosed which involves several steps, including enzyra-catalyzed reactions. "Previously, previous knowledge of the nature of these enzyme reactions was outlined

The invention relates to a method for isolating a specific genetic information containing nucleotide sequence, the synthesis of DNA with this specific nucleotide sequence and the transfer of the DNA into a host microorganism C

The invention is illustrated by the example of specific DNA sequencer transferred to a bacterium, namely the structural gene for rat pro-proinsulin, the gene for rat growth hormone and the human goitrogen hormone gene. It can be assumed that the method on the transfer of any DNA sequence of a higher organism? A higher organism is defined herein as any eucaryotic organism with differentiated tissues, including, for example , insects, molluscs, plants, such as a vertebrate, which is well applicable to any microorganism .

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zen, vertebrate, including mammals, to the latter cattle, pigs' primates and humans include "Under a microorganism refers to any microscopic living organism" of under the name Protist falls, where he procaryontiseh or eucaryontisch and In "game from a bacterium , P _r otozon can algae and fungi, including yeasts exist "is in the present method, a selected cell population, first using a new method isolated from the cells is extracted intact inRNS by a method bsi which virtually all RNase activity is suppressed," The intact messenger RNA from the extract is purified by column chromatography and then subjected to the action of reverse transcriptase, this action being in the presence of the four deoxynucleoside triphosphates required to synthesize a complementary (cDNA) strand. The product of this first reaction with reverse trans The remaining deoxynucleotide sequence, which is complementary to the parent mRNA, is incubated in a second reaction with reverse transcriptase or DNA polymerase in the presence of the four oeoxynucleoside triphosphates. "The resulting product is a double-stranded cDNA structure, the complementary strands are joined together at one end by a single-stranded loop "This product is then treated with lease for the specific Einzölstrang Nuc ~, which cleaves once rängige loop" the resulting double -cDMS is then extended 'by adding a specific DNA at both ends of the sequence Alner recognition or cleavage site of a _s Restriktionsanzyms having "the addition is catalyzed by sine ONS LigasQ" the extended CDMS is then reacted with

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treated with a rsstrictiono endonuclease, where self-complementary single-stranded 'ends' are formed at the 5 '' ends of each strand of the double helix

A Plasraid-DNA having a recognition site for the same restriction Ertdunuclease is treated with this enzyme _f urn cleaving the polynucleotide strand, and to generate self-kompleraentare once rängige Nuckleotidsequenzen at the 5 'ends "The 5 ^c -terminaleri phosphate groups on the einsträr, -gigen ends are removed to prevent the Piasroid forms a group capable of transforming a host cell ring structure "the ODNs _f prepared as described above and the plasmid DNA are then in the presence of DNA ligase incubated together" Under the described Resktionsbedingungen can the Eüldung a lebsn ^ enabled, only be closed ring of plasmid ~ DNS, if a segment of the cDNA is inserted "This Plasßiid the cDNA" containing sequence ivird because in © ine suitable WirtszelXe spent "cells _f who received a Viable ähiges ^ plasmid _f can be recognized by the appearance of colonies with a feature w contributed by the plasmid For example, drug resistance «Pure © bacterial strains. containing the recombinant .Plasmid with the built cDNA sequence, are then grown _t and the recombined plasmid is reisolated including In this way, large amounts are produced in recombinant plasmid DNA? from which one can deduce the specific cDNA sequence by endonucleation cleavage. can isolate the corresponding RestrUctionaenzym again "...

The basic method according to the invention is the isolation of any nucleotide sequence from a higher organism. "

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The method is useful for transferring a genetic code for a specific protein of medical or technical value and for the microbiological synthesis of such proteins. For the purpose of embodying the invention, the insulin encoding gene has been described Nucleotide sequence isolated from rat »transferred to and replicated in bacteria. The nucleotide sequence coding for rat growth hormone was isolated, transferred and replicated in bacteria. The method is based on the transfer of a nucleotide sequence _t isolated from human cell material, for example human insulin and Growth hormone or other polypeptide interferons applicable *

The present invention comprises a method for isolating a DNS'-Moieküis with specific nucleotide sequence and its transfer to a microorganism, wherein one back findete the original nucleotide sequence aar DNA according aer increase in the microorganism

The stages constituting the inventive method can be divided into four general categories?

I, The isolation of a desired cell population

There are two sources of a genetic sequence that encodes a specific protein. the DNA of the underlying organism itself and an RNA translation of aer DNA ₀ According to the current safety regulations of the National Institu-

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Tes of Health is mandated for the US that human genes of any kind can be introduced into recombined DNA only and then into bacteria after being cleaned very carefully, or when working in specially equipped (P4) facilities, see US Federal Register, BcU 41 _f No _e 131, 7 _e 7 _e 1967, S * 27S02 »27943β 3ed.8s A process of actual utility for the production of human protein, such as the present process, therefore, preferably works by isolating the specific mRNA which In this way, one has the further advantage that the mRNA can be purified more easily than DNA extracted from cells. also benefit from the fact that in highly differentiated organisms, such as vertebrates, it is possible to identify a specific cell population of specific localization in the organism whose function is mainly in the production of the protein in question development stage of the organism vorliegeru In such cell populations, a large proportion of the isolated from the cells of mRNA has the desired nucleotide sequence "the choice of the to be isolated cell population and the procedure used in the isolation can ira respect to the initial purity of the mRNA essential be *

In most tissues, glands, and organs, the cells are held together by an overall fibrous network of connective tissue that is primarily composed of collagen, but which may also contain other structural proteins, polysaccharides, and mineral deposits, as the case may be.

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ί

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The isolation of cells of a particular tissue necessarily involves methods of releasing the cells from the connective tissue matrix. The isolation and purification of a particular differentiated cell type thus involves two major stages of secreting the cells from the connective tissue and separating the desired cells from all others tissue types present in the tissue; The erf indungsgeinäB provided procedures are applicable "to the isolation of numerous cell types -from various tissues For example, the isolation of islets of Langerhans from the pancreas, the coding for the isolation of insulin raRNS is suitable are described _{"

Insulin-producing cells can also be derived from other sources, for example from the pancreas of. Fetal calf and from cultured islet tumor cells _e The isolation of pure islet cells is much easier in these cases, "especially when using reiner'Zellkulturen" In these cases, the procedure described above for the isolation of islet cells is not necessary "but said method is because of its general applicability advantageous"

Frequently, the amount of desired RNNS can be increased _t if one uses the Zsllrosktionen on stimuli from the environment correctly * Thus, for example, the treatment with a hormone can lead to increased production of the desired mRNA »Weiiere procedures, are breeding at a certain Temperature or in the presence of a specific nutrient or other chemical substance. In the isolation of rat i-growth hormone-rüRNS, treatment of cultured pituitary gland with thyroid

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hormone and glucocorticoids, the proportion of growth hormone "raRNS increased significantly in a synergistic manner

2 "Extraction of tnRNA

An essential feature of the present invention is the substantially complete removal aer RNase activity in the cell extract "The to be extracted mRNA 1st a single-stranded polynucleotides unpaired with a complementary strand * The hydrolytic splitting a single Phos" phodiesterbindung in Se.quenz would therefore total © make molecule for the purpose aer transferring an intact ge = netic sequence unusable in a microorganism "As already mentioned, the enzyme RNase widespread, active and exceptionally stable, it can be found on aer skin survives the usual rinsing of glassware and contaminated occasionally stocks organic chemicals "the difficulties are particularly acute when working with extracts of pancreas cells, since the thyroid is a source of digestive enzymes and therefore RNase rich _e the problem aer contamination by RNase arises in all wove en, una the presently disclosed process for the removal of the RNase activity is applicable to all tissue "The surprising effectiveness of the method is illustrated with egg successful isolation of intact mRNA from isolated pancreatic described home _seizollen

When on reißen.der cells and performing any procedure, the _t 'be used for isolating the protein substantially free of RNA, are employed erfindungsgemäS a combination of a chaotropic anion, a chaotropic cation

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- 19 -.

and cleaving a disulfide bond means an "The joint action of these agents is exemplified ihrer'Vervvendung in the isolation of virtually undamaged mRNA from isolated long Orhan Sechen islands from rat Pankreasbeschriehen * ·

The choice of suitable chaotropic ions is based on their solubility in aqueous media and their availability. Suitable chaotropic cations are, for example, guanidinium- * carbaraoylguanidinium-, guanylguanidinium, lithium ion and the like. Suitable chaotropic anions are, for example, the dodide, perchlorate, thiocyanates , Diiodo-salicylation and the like, "The efficacy of salts formed by the combination of such cations and anions depends, in part, on their solubility." For example, lithium diiodo-salicylate is a stronger denaturant than guanidinium-thiocyanate, but has one Solubility of only about 0.1 M, moreover, it is relatively expensive * The preferred combination of cation and anion is in the guanidinium thiooyanate _# because this salt is readily available and readily soluble in aqueous media, up to about Smolaren solutions _e

Thiol compound such as β-mercaptoethanol is known to cleave intramolecularly6 disulphide bonds in proteins by means of a thiol-disulphide-acid reaction, "numerous thiol compounds are effective in this direction, for example, β-merkeptoethanol dithiothritol, cysteine, propanone dimercaptan and the like "An essential characteristic is the solubility in water since the thiol compound must be present in large excess over the intramolecular disulfide _ä vm the exchange reaction -Perform to an end -to" ß-Merkaptoetha "nol is -because of its availability and cost

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Price preferred «.

In the inhibition of RNas'e aer during RNA extraction from cells or tissues, the effect of a chaotropic salt is dependent directly on its concentration, the preferred concentration is therefore the highest possible concentration · The success des'erfindungsgeraäßen method with regard to the preservation of intact mRNA during the extraction · Presumably due to aerobic denaturation of RNase and the extent of deriaturation »This may explain the superiority of guanidinium thionocyanate over the hydrochloride despite the fact. that the hydrochloride is only slightly weaker as a denaturant «. The effectiveness of a denaturing agent is defined as the threshold concentration required for the complete denaturing of a protein "On the other hand, the speed of the denaturation of many proteins depends on aer concentration of denaturing with respect to the threshold concentration _t increases from the fifth to ten," th power; compare Tanford * C _{0 c t} AdV ₁₅ Prot * Chenu 23, 12i (1968) _e This relationship shows qualitatively that only a slightly stronger denaturant than guanidine hydrochloride can denature a protein at the same concentration many times faster. "The Kinetic Relationships between RNase denaturing and mRNA preservation during its extraction from the Zellen.war to 2 _e of the invention itpunkt apparently not yet detected "the above considerations lead to it that the preferred Denaturierungsrnittel .wäre such that a low threshold concentration in combination with high Solubility in water "For this reason, guanidinium thiocyanate is preferred over lithium diiodo salate, although the latter is a compound

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stronger denaturant is because the solubility of guanidinium thiocyanate is much higher, and therefore it can be used at a concentration that allows faster RNase inactivation. Above considerations also explain the preference of guanidine thiocyanate over the comparatively soluble hydrochloride since the former is a slightly stronger denaturant "

The use of a disulfide bond-cleaving agent in combination with a denaturant enhances and enhances the action of the latter by thus fully unfolding the RNase molecule. "The thiol compound accelerates the progress of the denaturing process. by preventing a rapid Renaturierunq that can enter _t if the intramolecular disulfide bonds remain intact ^ .Femer is any, remaining in the mRNA preparation and this contaminating RNase virtually inactive in the absence of denaturant and Thiolf. In general, disulfide bond-cleaving agents with thiol groups are effective at any degree in the conformation, but a large excess of thiol groups is generally preferred over the intramolecular disulfide bonds for the equilibrium reaction to proceed in the direction of the olefin sulfide. On the other hand, many thiol compounds are malodorous and unpleasant high concentrations, so that in practice an upper concentration limit is formed · «. ER in e-Horkaptoethsnol "Concentrations reported in the range of 0.05 M to 1.0 M to be effective ,, and concentrations of 0.2 M in Oer isolation of non-degraded RNA from Ratienpankreas considered optimal."

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The pH of the medium during mRNA extraction from the cells may be in the range of pH 5 _f 0 to 8.0 "

After rupture of the cells, the RNA is secreted from the mass of cell protein and DNA. For this purpose, several methods are known, which are all suitable * A common practice is a precipitation method with ethanol in which the RNA is selectively precipitated. Preferably, the precipitation step is preferred bypassed and the Homogenisat directly on a 5 _f 7molare Casiuinchloridlösüng, which is located in a Z _e n trifugenröhrchen, piled up, whereupon. Centrifugation takes place; compare the description of glisine, V ", Crkvenjakov, R," and Byus, C _e -, Biochemistry 13, 2633 (1974). _c This method is preferred because it steadily maintains an environment unfavorable to RNsse, so that the RNA in high yield and free from DNA and protein «

The above procedures result in the purification of all RNA "from the cell homogenate." However, only a portion of this RNA is the desired mRNA. "To further purify this mRNA, one uses the fact that in the cells of higher organisms, ntRNA after transcription in the cell is further processed by bonding with polyadenylic _e Such mRNA having attached poly-Ä sequences can be selectively isolated by chromatography on columns with cellulose, is bound to the öligo thymidylate see Aviv, H * and leather, P ₉ IOCs cit ₀ the above processes provide substantially pure, intact mRNA from starting materials that are rich in RNase * 'the · purification of mRNA and the following carried out in vitro stages can with each mRNA. independently of their organism of origin _t be ausqeführt in virtually the same manner "

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Under certain conditions, _t, for example, in the use of cells from tissue cultures as a source of mRNA, the contamination with RNase may be so low that the above step a & r RNase inhibition is not necessary _e In these cases, the methods of the prior art can be used to decrease sufficient for RNasQ activity «

Figure 1 is a schematic representation of the remaining stages of the method, The first step is the formation of a DNA sequence that is complementary to the purified mRNA. The enzyme chosen for this reaction is reverse bridle riptase, although in principle any enzyme could be used that would form a complementary strand of DNA The reaction can be carried out under the known conditions using the mRNA as a template and a mixture of UBV four nucleoside triphosphates as precursors of the DNA strand. It is expedient for one of the deoxynucleic acid triphosphate G to be present in Position marked with 32 _p to follow the course of the reaction and to control the work-up and separation t, for example by chromatography and electrophoresis, and to be able to give quantitative yield data ί cf. Efetratis the _f A * et al, loc ₀ cit ₀ As can be seen from the drawing, As a product of the reaction with the reverse transcript, rnan receives a doppelst'rangj.go Ha arr.adolfonn with no * * · covalent bond between the RNA strand and your DNA strand «

Pas product of the reaction with the reverse trenocriotase

29 »8 _β 80 GZ 57 941 18

is obtained by standard methods from the Reaktionsgernisch used expedient - a combination of phenol extraction, chromatography on "Sephades G-LOO" (manufacturer Pharmacia Inc _ö Uppsala, Sweden) and precipitation with ethanol ^

Once the cDNA is enzymatically synthesized, the RNA template can be removed. Numerous methods of selectively degrading RNA in the presence of DNA are known. The preferred method is alkaline hydrolysis, which is highly selective and easily controlled by pH adjustment

After alkaline hydrolysis and subsequent neutralization, 32 _p- labeled cDNA can be concentrated by precipitation with ethanol / if desired »

The synthesis of a double-stranded hairpin-shaped cDNA is carried out using the corresponding enzyme _t, for example with DNA polymerase or reverse transcriptase.

The reaction conditions are similar to that described above, including the use of a labeled in ec-position with 32p nucleoside triphosphate, "Reverse transcriptase is available from various sources, for example from Bird Hyeloblastosisvirus (the virus is available from the Life Sciences Incorporated» St. Petersburg *, which produces it under a contract with the National Institutes of Health)

After the formation of the cDNA hairpin it may be recommended

29. 8 _O 80

GZ 57 941 18

pc "

be _f to win the DMS from the purely Resktlonsgemisch * Again, it is advantageous to use _e phenol extraction chromatography on "Sephatex G-IOO" and ethanol precipitation to purify the DNA product and exemption from contaminating protein "

The hairpin structure may be converted into a conventional doppolsträngige DNS _£ by _t the once-ranking loop connecting the ends of complementary strands, away "To this end, various enzymes for disposal, which cause a specific hydrolytic cleavage of single-stranded DNS'areas" A for this purpose suitable enzyme is the SI Nuclesse from Aspergillus oryzae (the enzyme can _f obtained from Miles Research Products, Elkhart, Indiana Vs? ground) e treatment of hairpin-shaped DNA with Si 'nuclease leads to high yields of cDNA Molekülan with bass pair ends «. Extraction _s chromatography und'Ausfällung with ethanol are carried out as described above. " The use of reverse transcriptase and Sl nuclease in the synthesis of double-stranded cDNA transfers of mRNA would described by Efstratiadis, et al _e "loc" cit _e "

Optionally, one can increase dan proportion of cDNA molecules with blunt ends by treatment with E _s coli DNA Polymeras © I in the presence o & r four öeoxynucleosid triphosphates "Di © combination of exonuclease and polymerase activity dos enzyme leads to removal of all stand out η d β r 3 '· * E η de η u η d to the e ff ect η s of all h θ r ν ο pending 5'-Enderic This lV ₈ ise is due to the participation of the bulk of the cDNA molecules the following linkage reactions.

29 * 8 _β 80 GZ 57 941 IS

The next step in the process is to control the ends of the cDNA product such that there are suitable sequences at each end which have a recognition site for a restriction endonuclease. _Β The choice of DNA fragment to be added to the ends is determined by convenience in U.S. Pat handling, attached to the ends to be added sequence restriction endonuclease is selected according to the chosen, and this choice is again based on aer choice of DNA vector to which the cDNA is to rekorabinieren "the selected plasmid should possess at least one point which For example, the plasmid pMB9 has a recognition site for the enzyme Hind IH ₆ Hind III is isolated from Hemophilus influenza and purified according to the method of Smith, H "0 and Wilcox," IV * Mol "Biol * 51, 379 (1970) purified" The hemophilus aegyptious Hae III enzyme is made according to the method of Middleton, D _e H, 'Edgell, M * H _e and Hutchinson-III, C. A * D _e Viroi' 10, 42 (1972). "An enzyme from Hemophilus suis, called Hsu I, catalyzes hydrolysis at the same recognition sites as Hind HI _6. These two enzymes are therefore considered to be functionally interchangeable."

It is convenient to use a chemically synthesized dop ™ pel-stranded deoanucleotide having the Hind III recognition sequence for attachment to the ends of the oDNS ~ Popp 3 'strand _e. The double-stranded decanucleotide has the sequence shown in Figure 1, see Heyneker, H _e L * st al « and Scheller, R _'e H''et al, "loc" cit "Various such synthetic sequences with recognition sites are now available, so that one can prepare the ends of the double DNA so that they suf di © action of one of the zahlrei" respond to restriction endonucleases,

29 _β 8 _β 80 GZ 57 943, 18

The binding of the recognition site sequence to the ends of the cDNA can be accomplished in a known way. The method of choice is a reaction called "blunt-end linkage" which is catalyzed by a DNA ligase prepared by the method of Panet, A * et al _"t Biochemistry 12, 5045 (1973) was purified" The reaction Oer link blunt ends was by Sgaramella, V ₆ et al _"t loc * cite described The product aer linking a cDNA with blunt ends with a large molar excess a Double-Stranded Qecanucleotide Having a Hind III Endonuclease Recognition Site is a cDNA Having This Hind III "Recognition Sequence on Each End" Treatment of the reaction product with Hind III endonuclease results in cleavage at the recognition site and formation of single-stranded self-complementary 5 ^f - Ends, see Fig _t 1 *

4 * Inventive Formation of a Recombinant DNA »Trans ~

In principle, a variety of viral and plasmid DNA can be used for recombination with the cONS prepared as described above. "The basic requirements are; the DNA transfer vector must be capable of entering a host cell, replicated in the host cell, and also possessing a genetic confinement size; which allows those Wirtszsllsn, which started production n vector, etc. and off'zusondern "For reasons of public safety Ausvvahlbereich should be limited to those Transför-Vektorsrten who try to jeweiligoi suitable he" appear, corresponding to the above-mentioned guidelines

^2S * ^{8β 80}

the National Institutes of Health * The list of approved DNA transfer vectors is constantly growing "as new vectors developed and approved by the relevant committee of the National Institutes of Health _e According to the invention, of course, provided the use of any virus and Piasmid DNA which the skills described have, including those whose approval is yet to come "Suitable transfer vectors _t approved for use today are various derivatives of bacteriophage y ^ (see ο eg Blattner, F * R _e, Williams, B ₀ G *, Blechl , A "E *, Denniston-Thompspn,! << =, Faber _s Η _β Ε" , Furlong, Le A *, Grundwald, D ₉ D _e , Kiefer, D "0 _e , Moore, 'D» O _e , Schumm, O * W _0, Sheldon, Ec L "and Smithies, Sun, Science 196, 161 (1977)) and derivatives of the plasmid col El (cf. _c, for example, Rodriguez, R _c L *, Bolivar, S ,, Goodman, H * M ₀ , Boyer, H _e W. and Betlach * MN, ICN-UcIa Symposium on Molecular Mechnisrous In The Control of Gene Expression, D ", P _e Nierlich, W _e D _et Kutter," F _e , Fox, Eds "(Academic Press, NY, 1975) S * 471. 4-77) β Plasmids from col El are relatively small, have molecular weights on the order of less than millions, and have the property of increasing the number of copies of plasmid DNA per host cell from 20 to 40 under normal conditions to 1000 or more if you can do that. Host cell with chloramphenicol treated, the ability to increase the gene amount in the VVirtszelle allows the host "to cause cell that it produces mainly proteins encoded by the genes on the plasmid under certain Urnständen, under the control of the experimenter _β Such derivatives of col El are therefore preferred transfer vectors according to the invention. Suitable derivatives of col El include the plasmids pMB-9 which contain the gene for tetracycline resistance.

29. 8 * 80 GZ 57 941 18

stenz, and pBR-313, pBR-315, pBR-316 ', pBR-317 and pBR-322, which possess a gene for ampicillin resistance in addition to the tetracycline resistance gene. The presence of drug resistance genes is a convenient way to select the cells that have been successfully infested by the plasmid, as colonies of these cells grow in the presence of the drugs, while cells without the plasmid do not grow or colonize * 3 In the examples of this description, a plasmid was always used from col El, which in addition to the drug resistance gene possessed a Hind III recognition site * '

As in the case of the plasmid, the selection of the appropriate host can be done from a very broad spectrum, but the area has been narrowly restricted for reasons of public safety. " E "coli strain with the designation X-1776 was developed and approved by the National Institutes of Health for the method described above where P2 devices are prescribed ι compare Curtiss, 111, R _'; Ann "Rev" Microbiol *, 30, 507 (1976) * E ₆ coli RR-I can be used when PS facilities are available _o As in the case of Plaomide the invention provides, of course, the use of strains of any host cells from which to , Harboring of the chosen vector, including protists and other bacteria , for example yeasts

Recombinant plasmids are obtained by mixing Restrilctions'ndnduclease-treated plasmid DNA with cDNA, both containing analogously treated end groups. To the possibility that cD.NS-Seomente combinations

29 * 9, 80 224 26 / · GZ 57 941 · 18

The plasmid DNA is used in a molar excess via the cDNA. This method of 'has hitherto' led to the fact that the majority of the plasraids circulate without an inserted cDNA fragment. The subsequently transformed cells then contain mainly plasmid and not recombined with cDNA plasmids "the result is a very tedious and time-consuming Se ~ lesson process _a according to the prior aer art, this problem has been solved _t by trying to produce DNA vectors with a recognition site for the restriction endonuclease in the middle a suitable marker gene, such that insertion of the cDNA _t divides the gene "whereby loss occurs UBr encoded by the gene function"

Preferably, a method for decreasing the colony count * to be assayed on recombined plasmids is used. Treating plasmid DNA cleaved with the restriction endonuclease with alkaline phosphatase (commercial enzyme) is treated by treatment with alkaline phosphatase the 5 '~ terminal phosphate groups are removed from the produced with aer endonuclease ends of the plasmid and the self-join of the plasmid DHS is prevented the ring formation and transformation hän "<thus gen on the insertion of a DNA fragment containing 5' ~ phosphorylated ends This procedure reduces the relative frequency of transformations without recombination to less than 1 to 10 *.

The process of the present invention is based on the fact that the DNA ligase-catalyzed reaction occurs between an S'-phosphate 'DNA end group and an S'-hydroxylamine-DNA end group. When the terminal 5'-phosphate group is deoxygenated'

29 "8" 80 GZ 57 941 3.8

ferrite, no binding reaction occurs «Is double-stranded DNA to connect, so are two situations possible, shown in Table 1?

Table 1

case

III

Reaction participant 3 ·

OH ^H 2 ° 3 ^P0

· - »OPO_H"

3 ¹

-OH.

5 ^! 3 '

OH

5 ^s

OH

HO

Ο-ίΛωΓΪ

Ligase product

-P-d-

5 '

3 '

-0-P "0-

5 ¹

OHH O

I CC

5 '3'

no reaction

- ^{+ H} 2 °

Xn iabolle Ϊ the double-stranded DNA is represented diagrammatically by two parallel lines, with the 5 'and 3''endgroups' depending on the hydroxyl or phosphate groups. ₅ In Fail I, S'-phosphine groups are present on both reactive end groups before, with deta result that both strands werdsru covalently bound in the fur Il has only one of the strands to be connected to a terminal 5 ⁶ ~ Phcsphatgruppe with the result that * 'is a covalent bond einsirängige

29 «8 * 80 GZ 57 941 18

The non-covalently linked strand remains bound to the molecule in a known manner due to hydrogen bonding between complementary base pairs on the opposite strands. In case III, none of the ends of the reactants has a 5'-P ^ csphatgruppe so that no binding reaction can occur "

Undesirable © binding reactions can therefore been prevented by treating corresponding ends of the reactants, the compound is to prevent, with removal oer 5 * phosphate groups _e It can be any method for the removal of 5 '' phosphate groups apply that the DNS is not damaged otherwise The hydrolysis catalyzed by the enzyme alkaline phosphatase is preferred, »

The method described above is then useful * when a linear DNA molecule is to be cleaved into two subfragments, typically with a restriction endonuclease, and then reconstituted in the original sequence. "The subfragments 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 linkage of DNA fragments, can be used for this purpose, see Sgara ™ meila, V", _f van de Sande, 0, "H _e and Khorana, H _e G," Proc "Nfäti, -Be" USA 67, 1468 (1970) · Are the sequences to be joined not dull, it can be the ligase coli obtained from e ₆ use * compare Modrich, P « and Lehman ,, I * R ₆ * CJ ₀ Biol, Ehern« 245, 3626 (1970) «

The reconstitution of the original sequence by rescri

29 «9« 80 GZ 57 941 13

iion endonuclease is greatly improved when using a method that prevents reconstitution in the wrong sequence. This is done by treating the cDNA fragment of homogeneous length of the desired sequence with an agent containing the 5 '-' terminal phosphate groups of the cDNA removed prior to cleavage of the homogeneous cDNA with a restriction endonuclease © Man again preferably uses alkaline phosphatase _. The S'-terminal phosphate groups are a structural prerequisite for the subsequent binding action of DNA ligase, which allows for the reconstitution of the cleaved. Subfragments are used "Ends without 5 '-' terminal phosphate group can therefore not be covalently bound. The DNA subfusions can only be bound to such ends having a 5-phosphate group.

This method prevents the most important undesired binding, namely the connection of two fragments with reverse ssquens, called "an-front _f instead of front". "Other possible side reactions such as dimerization and cyclization are not prevented." as they take place in a reaction vora type II in table I "these adverse reactions are less detrimental _s because they lead to physically separable and identifizierberen products" which is not the in Naukumbination in the wrong direction. Fsii lsi «

To illustrate the method described above rat insulin has been isolated, and cDNA encoding recombined with a Plsomid The DNS Molekülo were used for transformation of E ₈ coli X-1776, the selection of the to -transformierenden cells was made by growth on a

β OAf-. 29. 9 * 80

GZ 57 941 18

, - 34

tetracycline-medium ".A obtained from transformed cells recombined plasmid" DNA contained an inserted DNA fragment of about 410 nucleotides in length _{o f} More Neukorabinationen had been isolated in the same manner were also analyzed The inserted fragments were digested with Hind-LLI or HSU The DNA sequence was released from the plasmid and the DNA sequence was determined according to the method of Maxam, Ae M ₆ and Gilbert, W ₄ , Proc _e Nati * Acad · Sei USA, 74, 560 (1977). " The nucleotide sequences of the inserted DNA fragments were overlapped and contained the entire coding reaction for rat proinsulin Ϊ and 13 out of 23 amino acids of the pre-peptide sequence. A compilation of the nucleotide sequence of this region is shown below.

On similar. As was growth hormone from rat encoding cDNA isolated, recombined with a plasmid and transferred to Eecoli "After extensive replication in E ₀ coli a nucleotide sequence of about 800 nucleotides in length was isolated, the entire coding sequence for rat growth hormone, and parts of the Vorlauferpeptids and Part of the 5'-untranslated region contained ₉

The process described is generally applicable to the isolation and purification of a gene of a higher organism, including human genes _e whose transfer into a microorganism and replication in the micro Organismuse Also, new recombinant plasmids., Which are all or contain a portion of the isolated gene as described Furthermore, hitherto unknown new microorganisms are described which contain, as part of their genetic make-up, a gene of a higher organism

^{29β 8} * ^so

GZ 57 941 18

- 35

Examples describe in detail the process in the application _s of the isolation, purification and transfer of Rescue-Insulingon in E _c col: 'u In the examples, recombinant plasmids, the parts of rats are further characterized, ~ Xnsulingen, rat iVachstumshormongen, human Insulingen and human growth hormone gene included, as well as the said genes containing new microorganisms e.

Example 1

For the suppression of purified rat islet cells, the pancreas of an anesthetized rat is infused with Hank's saline solution, retrograde infusion into the pancreatic duct. Hank's saline solution is a known and commercially available solution Hankscher solution of OC minced and digested with collagenase and trypsin inhibitor "All procedures are carried out at 0 to 4 C, unless otherwise stated" The conditions during digestion are extremely critical * Two crushed pancreas in 8 ml total volume Hankscher solution are in a 30 -ml glass tube filled «All glass parts were treated with silicone (trade name Silicladj manufacturer Clay-Adama Division, Becton-Dickinson rnc« _t Parsippsny * New Dergey) The incubation mixture contained 12 mg of collagenesis an enzyme obtained from Clostridium histolyticum (prepared essentially according to the method of Mandl _f ~ X ₍ ,, Maakeaan * α * D _e and .Howes. E ₀ L * _t CJ _e ClIn-T. η vest * 32 ₁ 132.3 (1943) ~ type CLS IV, manufacturer · Worthington Biochemical Corporation, Freehold, New Oersey) and 1 mg of soybean "trypsin inhibitor".

: 29 "8 _β 80

#. GZ 57 941

36 -

tor, manufactured by Sigma Chemical Company, St _4, Louis, Missouri "Incubation was bei.37 ⁰ C for 25 Hin" shaking of 90 movements per minute, "Constant monitoring was needed to ensure that the collagenase digestion in optimum extent proceeded _β too short incubation, the islets were incomplete release, is too long incubation begins its decomposition, * After the incubation, the glass tube was 1 min "at 200 χ G centrifuged" the supernatant liquid was decanted, and the cake was washed with Hank's solution , this operation was repeated five times * After the last centrifugation, the cake (manufactured by Pharmacia Chemical Company, Uppsala, Sweden) was dissolved in 15 rnl of Hsnüolsprodukts Ficoll with a density of 1.085 suspended a layer of 8 ml Ficoll density 1.080 was added, and a layer of 5 ml Ficoll of density 1.060, then the glass tube in a swinging cup rotor 5 min 500 χ G and then 5 min _s at 2000 χ G centrifuged The acinar cells remained on the ground, the islet cells rose to the gradient and formed a layer between the two upper classes * The islet cell layer contained contaminating ganglion cells, lymph nodes and connective tissue "Big contamination fragments were forward The remainder of the preparation was removed under the microscope by hand from visible impurities using a micropipette. The cell preparation was then diluted in Hank's solution and centrifuged. The supernatant was decanted off and the cell cake was stored frozen in liquid nitrogen.

The islet cells fused together by 200 rats were dissolved in 4 molar guanidinium thiocyanate solution (trade name

29έ 8 _β 80

£ 4 Ä..Ö /. , GZ 57 941 18

· - 37 -

Tridom, manufacturer? Fluka AG, Chemische Fabrik, Buchs) the Imolar buffered to beta-mercaptoethanol and adjusted to pH 5.0, at 4 ⁰ C. The homogenate was homogenized 1.2 ml 5,7molare cesium chloride solution containing 100 mM ethylenediaminetetraacetic acid overlaid, and 18 h "at 37 revolutions / Hinute in the rotor SW 50.1 a Beckman ultracentrifuge at 15 ⁰ C centrifuged to the bottom of centrifuge tube *

centrifuge centrifuged at 15 ⁰ C. While migrating the RNA

Polysdenylated RNA was isolated by chromatographing the total RNA preparation of oligo (dT) cellulose by the method of Aviv, H *, and Leder, P "loc" cit *

For transcription of total polyadenylated RNA from the islets of Langerhans of rats in cDNA reverse was transcriptase from avian myeloblastosis virus (obtained from Life Science Inc _c St ₀ Petersburg, Florida) is used, "Reactions were performed in 50 mM Tris-HGL, pH 8.3, 9 mM MgGIp, 30 mM Nagi, 20 mM .beta.-mercaptoethanol, 1 mM each of 3 non-radioactive deoxyribonucleoside triphosphates, 250, uM of the fourth, labeled in position cg 32 _p deoxynucleoside specific Triphoephatj Activity 50 to 200 Curie / mole _f 20 μg / ml Gligo = dT. ,, _, _ ,. ρ (manufacturer Collaborative Research, S-VaItham, Massachusetts), 100 ug / rnl polyadenylated RNA and 200 Einheiien / ml reverse transcriptase performed * The Gsmisch was 15 min * at 45 ⁰ C incubated _ö After addition of EDTA-NA ₀ bie 25 mil .wurde the solution with an equal volume of water gesattigtom phenol extracted, then the aqueous Phaso was purified on a gofülltsn with Sepbadex-G-IOO column of 0.3 crn diameter and 10 cui height in iO-mM Tris-HCl, pH 9, 0, 100 mM NaCl ₉ .2 mM EDTA. Chromatographed * The eluted nucleic acid was precipitated with ethanol after addition of ammonium acetate (pH 6 _f 0 to 0 _e 25 M),

29, 8 _β 80 GZ 57 941 18

The precipitate was centrifuged, the cake was in 50 / ul of freshly prepared 0! Solved molar sodium hydroxide solution and 20 minutes' incubation at 70 ° C to the RNS to hydrolyze _e The mixture was with! Molar sodium acetate, pH 4.5, neutralized and the 32p ^ cDNA product was precipitated with ethanol and redissolved in water, "sample" a stranded cDNA were on native polyacrylamide gel by the method of Dingman _s C ₈ W _e and Peacock, Α "C _t, Biochemistry 7 , 659 (1958). The gels were dried and the 32 _p -DNA was obtained by autoradiography using an i-odak-i-o-Screen ~ NS-2T film 8 (manufactured by Eastman Kodak Corp., Rochester, Conn . determined New York) »the cDNA was heterodisperse, as shown in the electrophoresis" It contained at least one major cDNA of approximately 450 nucleotides (comparison with known standards) *

The single-stranded cDNA obtained according to Example 1 was treated with reverse transcriptase to prepare the complementary strand. The reaction mixture contained 50 mM Trel HCl, pH .8.3, 9 mM MgCl ₂ .10 mM dithiothreitol, 50 mM in each case three non-labeled deoxyribonucleoside triphosphates * i-mM of a 32 _p- labeled nucleoside triphosphate with the specific effect of 1 to 10 Curie / 1 lira oil, 50 / μg / ml cDNA and 220 units / ml Reverse transcriptase _e the reaction mixture was "120 min" at 45 ⁰ C incubation "the reaction was stopped by adding EOTA Nap to 25 mM, then extracted with phenol, chromatographed on Sephadex G-IOO and precipitated with ethanol A sample of Reaction product from 500 to 1000 oprn was determined by gel electrophoresis as described in Example i,

£ L * £ 4. & a

GZ 57 941 18

"Heterodisperse bands" centered around 450 nucleotides in length were observed when compared to Standard Proberu Pr.ob'en of the DNA reaction products of Examples i and 2 were treated separately with excess Hae III restriction endonuclease and analyzed by gel electrophoresis * Both products were cleaved by the endonuclease so that two bands of radioactivity were observed in the gel electrophoresis "resulting from the cleavage of the double stranded cDNA binding represented cleavage products of essentially the same chain length as in the cleavage of single-stranded cDNSo

The double-stranded reaction product of Example 2 was dissolved in a concentration of 2-5 μg / ml with 30 units of Sl nuclease having an activity of 1200 units / ml (manufacturer J Milec Laboratories, Elkhart, Indiana) in 0.05 M Sodium acetate, pH 4.6, 0 _f 3 H sodium chloride, 4.5 mM ZnCl _? Incubated for 30 min at 22 ⁰ C _e and a further 15 minutes' at 10 C _e With the addition of trie base to 0.1 M final concentration, EDTA to .25 mM and E * coli tRNA (prepared by the method of Ehrsnstein, G ", Methods in Enzymology, eds" Colowicki S _s P "and Kaplan, N ₄ O _ef BCU izk, S _e 588 (1957)) to 40 ^ ug / digestion ml'wurde stopped" After extraction dos ReaktionsQQmioclieü m- if phenol and Chrcmatographieren on Sephadex G-IOO the eluted · 32. ~, -cdns Re with ethanol was e.usgefälltO This treatment resulted in a high yield cDNA molecules with en basengopasrtcn ends Oer for linking blunt ends required to chemically synthesized Decanucleotide were HindllX decamers

29 »p. 80 GZ 57 941 18

were prepared according to the method of Scheller, R _e H ", Dickerson, R * E *, Boyer, H" .W ", Rigge, A" D * and Itakura,! <"Science 196, 177 (1977) the Hind III decsmere with the cDNA was made by incubating at 14 C in 66 mM Tris-HCl, pH 7.6 _f -6.6 mM MgCl 2, 1 mM ATP ₄ 10 mM

5 dithiothreitol, with 3 mM Hind III decamers at 10 cpm / pmole and T4 DNA ligase, about 500 units / milliliter, for 1 hour. The reaction mixture was then warmed to 65 ° C for 5 min Inactivate «Then KCl were up to 50 mti. Endkonzontration, ß-mercaptoethanol to 1 mM final concentration and EDTA admit to 0.1 in M final concentration prior to the digestion with 150 units / ml, which was 'carried out for 2 hours at 37 ⁰ C' Hind III and Hae III ~ ~ Endonuc- 'lease are commercial preparations available from New England Bio-Labs, Beverly, Massachusetts * the reaction product was purified by gel electrophoresis as described in example 1, analyzed "Here, a peak was observed, which corresponded to a sequence of about 450 nucleotides, also fragments of the cleaved Hind III deca »mers were observed«. ^ _-

Example 4

Plasmid DNA pM3-9 ~ (preparation see _e Rodrigues, R ".L _e, BoIiver, F _0, Goodman, H * M," Boyer, H "Vt / _β and Betlach, M _0, ICN-UCLA Symposium on Molecular and Cellular Biology, Wiarlich, D _e .P ",. Rutter, W" 0 _s, and Fox, C _s F _{e t} Eds '' (Academic Press, New York 1976) S _c 471-477) was at the Hind- III-recognition site cleaved with Hsu-l-i-endonuclease, then treated with alkaline phosphatase (type BAPF _f V / orthington Biochemical Corp., Freehold, New Dersey)

29 * 8 _e 80

GZ 57 941 18

~ 41

Enzyme was present in the reaction mixture in an amount of 0.1 unit / microgram of DNA, the reaction mixture was incubated in 25 mM Tris-HCl for pH 8 for 30 minutes at 25 ^° C. ₍ then phenol extraction to remove the phosphatase) Precipitation with ethanol was added to the plasmid DNA treated with phosphates to cDNA which had Hind III cohesive ends in a mole ratio of 3 moles of plasmid to 1 well. cDNA _e The mixture was in 66 mM Tris, pH 7.6, 6 _f 6 mM MgCl _2, .10 mM dithiothreitol and 1 roM ATP X h 'at 14 ° C incubated Liqase "

X hrs at 14 C in the presence of 50 units / ml T4 DNA

Then, the reaction mixture was added directly to a suspension of Ee coli X ··· 1776 cells which were prepared for separation as follows: The cells were grown to a cell density of about 2 × 10 4 cells / ml in 50 ml of medium. 10 g / l tryptone, 5 g / l yeast extract, 10 g / i sodium chloride "2 mM NaOH, 100 μg / ml diaminopimelic acid" and 40 μg / ml thymine, and 37 ° C shows γ, cells were incubated by stirring centrifugation at 5000 χ G at 5 C gesammol, _e t 'suspended chloride solution, centrifuged again and resuspended in 20 ml transformation buffer containing 75 mM CaCl _2, 140 mM NaCl and 10 Jnn Tris pH 7 in 20 ml of cold lOraillirnolarer sodium " 5 contained, and 5 min _e stehengelasnöiu -in ice the cells were then centrifuged and resuspended in 0.5 rolling transformation buffer Di © transformation is "" by mixing iOO yul the cell suspension with 50, ul of recombinant DNA (Yug .1 / ml ) '* Dos mixture became 15 miru at 0 ⁰ C incubated _? then 4 min * treated at 25 ⁰ C and 30 min "boi 0 ⁰ C. The cells were then transferred to agar plates for growth under selected conditions *

'9 9 3 947 ·' ^{29ί 8} · ⁸⁰

The Choosing the recombinant plasmids was carried out with δ micrograms / ml tetracycline at transformation with the Hind "IU product" has been a- called pAU-1 Neukombip.ation isolated Crude plasmid preparations with 2, ug to 5, .ug DNS ( isolated from pAU-1) were treated with excess Hsu-I endonuclease. Then 10mM EDTA-Na ₂ and 10 % (w / v) cane sugar were added and the mixture was digested to 8 % polyacrylamide gel In a similar experiment, the transfer vector used was the plasmid pBR-322. All conditions remained as described except that the last selection of the recombinant clones was on 20 μg / ml ampicillin-containing plates.

After elution from the gel, DNA was labeled by incubation with Ί -P-ATP and the enzyme polynucleotide kinase under the conditions described by Maxam and Gilbert loc _e cito. "The enzyme catalyzes the transfer of a radioactive phosphotro group of ι- ⁴ ^ P ATP on the 5 ¹ -terminals of the DNAs The enzyme was from E. coli according to the method of Panet, A, et al. Biochemistry 12, 5045 (1973). The DNA thus labeled was digested with Hae III endonucleass according to the protocol of Example 2, and two labeled fragments of about 265 and * 135 base pairs, respectively, were prepared under the conditions described in Example 1 The isolated fragments were subjected to specific cleavage reactions, then the sequence analysis was carried out according to the method of Haxam Gilbert, loc * cit * The sequence of the following Table 2

on and

29 «8» 80 GZ 57 941 18

- 43

draws on a compilation of the results of these test series and similar series of experiments with cDNA and. Plasmid vectors such as ccd El PMSS-9 and pBR 322 ~ _e at the 5 'end is undetermined a sequence of estimated 50 bie 120 nucleotides in length, and the poly-dA-segment at the 3' end is of different length * These Sequence corresponds to the current state of knowledge, whereby, of course, further investigations may open up additional details or may necessitate a slight revision of some areas. The corresponding amino acid sequence of rat proinsulin begins with the triplet position designated 1 and ends with 86 designated triplet position,. Some uncertainty remains with respect to the dashed line underlined sequence *

⁸⁰

041 18

- 44

Table 2

/ indeterminate / --GCC CTG CTC CTC TGC GAG CCC AAG CCT '

20

GGT CCT CAC CTG GTG GAC GCT CTG TAG CtG GTG TGT CCG

40 GAA GTG GAG GAC CCG CAA GTG CCA CAA CTG GAG CTC GCT

60. 70

CTG GAG GTT GCC CGG CAG AAG CGT CGC ATT GTG GAT CAC

86 AAC TAC TGC AAC TGA GTTCAATCAATTCCCGATCCACCCCTCTGCA

ii: dashed line underlined ~ areas of existing ones

uncertainty

.29 «8« GZ 57 941

(Continuation)

1 CCT CAG CCT TTT GTC AAA CAC CAC CTT TGT

30

GAA CGT GGT TTC TTC TAC AGA CCC AAG-TCC CGT CGT

50 CGAGGC CCG CAG GCC GCG CAG CTT CAG ACC TGG CCA

80 TGC TGC ACC AGC ATC TGC TCC CTC TAG CAA CTG GAG

TGAATAAACCCTTTGAATGACG-poly A

29 * Se 80 GZ 57 941 18

A nucleotide sequence encoding a human insulin is isolated, purified and inserted into a plasmid according to the protocol of Examples 1 to 4, using. emanating from a suitable source, for example, a donated pancreas or a fresh corpse or a human insulinoma. "A microorganism is produced essentially according to the protocol of Example 4 , with one the A and B chains of human Insulin-encoding nucleotide sequence »The known amino acid sequence of the Α chain is t

1 10

Giy ^ Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr-Glu-

20; Leu "Glu-Asn-Tyr-Cys-Asn

The amino acid sequence of chain B is?

1 oo

Phe-Val-Asn-Glu-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala ^ Leu-

20 30

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

The Arainossuresequenzen be quantified starting from the end with free Arninogruppe, see Smith, L ₈ F ^, Diabetes 21, (Erg _e Z) ₁ 458 (1972) *

Beisplay 7

For genes of non-human origin, the US's own rheumatoid arthritis does not require the isolation of a high purity cDNA gene as with human cDNA, therefore it may not be the whole rat growth hormone structural gene

, 29 · 8 «80. # / GZ 57 941 18 ·

Sas «β * Ii

- 47 -

to isolate cDNA containing electrophoretically isolated DNA of the expected length of about 800 base pairs (corresponding to the known amino acid length of the growth hormone) isolated. As a source of rat growth hormone mRNA, cultured rat pituitary cells (subclone of the cell strain GH-I, available from the American Type Culture Collection) were used, see Tashjian, A ₉ H et al ₉ , Endochrinology 82, 342 (1968). When these cells are grown under normal conditions, the growth hormone mRNA constitutes only 1 to 3 % of the total poly A-containing RNA. However, the amount of growth hormone mRNA was increased by the amount of other m Rli S in the cell due to the synergistic action Thyroid hormone and glucocorticoid RNA The RNA was obtained from 5x10 cells grown in a suspension culture and predisposed to growth hormone production by adding 1 mM dexamethasone and 10 mM L-triiodothyronine to the medium 4 days prior to collection Lines "The polyadenylated RNA was isolated from the cytoplasmic nuclear fraction of the cultured cells, see Martial, 3 * Α., Baxter, 3" D, ", Goodman, H * Ho, and Seeburg , P ₄ H," Proc Nat " Acad, "USA 74, 1816 (1977), and Bancroft, F _e C", Wu ₅ "G * and Zubay, G ₀ , Proc _s Nat * Acade Sei", USA 70, 3646 (: 1973) _e The rnRNAs were then essentially in accordance with the instructions of Examples 1, 2 u nd 3 further purified and transcribed into double-stranded cDNA after the fractionation of gel electrophoresis has been a weak but distinct band corresponding to a DNS. with about 800 base pairs in length

The treatment of the whole, obtained by transcription from the KrMS the cultured pituitary cells CDMS with .Hhal-Endonuclea.se gave (after te ragmen two DMS Hauptf elsk-

29 * 8 «80 GZ 57 941 18

- 43 -

trophoretic separation) corresponding to about 320 nucleotides (fragment A) and 240 nucleotides (fragment B). The Nucleotidsequenzanalysa of the fragments A and B according to the procedure in example 5 revealed that these fragments were actually portions of the coding region for rat growth hormone, and on the basis of the known amino acid sequence for rat growth hormone, and by comparison with other known IVachsturnshormonsequenzen compare ι Wallis, Mp and Davies, 'R _e V' N *, Growt Hormones and Related Peptides (Ed. Copecile, A _e and Muller E _e E ₃ ), S 1 - 14 (Elsevier, New York, 1976), and Dayhoff, M. 0 _e, Atlas of protein Sequence and Strukture, 5, Erg * 2, S, 120-121 (National Biomedical Research Foundation, Washington, D 'C, 1976) * If the double-CONS 800 base pairs of the above treated electrophoretic isolation described analogously with Hha I endonuclease, as obtained under the main degradation products of two fragments which correspond in length to the fragments A and B *

Since the rat Waehstumshorrnon cDNA was not cleaned with about 800 base pairs with recourse to · the treatment with restriction endonuclease, the DNA had to the purpose of removing unpaired single-stranded ends treated vverden _e In practice -Removes these unpaired ends before aer electrophoretic separation in 25, μl 60 ml A Tris-HCl, pH 7.5, 8 mM MgCl ₉ , 10 mH β-merkeptoothanol, 1 mM ATP and 200 μM of each of the nucleotides dATP, dTTP, dGTP and dCTP _e The mixture was combined with one unit C _rt CCLI DNA polymerase I incubated at 10 C for 10 minutes, "to remove any protruding 3 ¹ tails and all protruding ^5! -During the end of the "DNA polymarase I" is a commercially available product (βpehringer-Mannheim Bicchamicals, Indianapolis, Indiana) ".

β,

29 »8 * 80 GZ 57 941 18

- 49 -

Chemically synthesized Hind IIl linkers were added according to the protocol of Example 3 to the approximately 800 base pair rat growth hormone cDNA. The plasmid pBR-322 with an ampicillin resistance gene and a single Hind III site in the tetracycline resistance gene was labeled with Hind III endonuclease and alkaline phosphatase according to the procedure of example 4 pretreated "the thus treated plasmid was oer rat wax Tunis hormone cDNA with the 800 bases" combined pairs in a DNA ligase reaction according to example 3 * the mixture of ligase reaction was to Transfermierung a suspension of e coli _e ~ ~ ~ X: L776 cells that had been treated according to example 3, "used the re ~ colonies were combined due to their growth containing ampicillin nutrient plates and their inability to grow on 20 μg / ml tetracycline secreted »10 of such colonies were obtained containing the plasmid with an insertion of 800 base pairs« Die Fre was done by Hind III split «

The 800 base pair rat growth hormone DNA (RGH DNA) was isolated in preparative amounts from the recombinant clone pRGH-1 and the nucleotide sequence was determined as described in Example 5. The nucleotide sequence comprised portions of the 5 ° rat growth hormone untranslated region and a sequence of 26 amino acids found in the growth hormone protein precursor prior to secretion »The mRNA sequence deduced from the gene sequence. sequence shows the following Table 3 _e The predicted amino acid sequence agrees well (except for the positions 1 and 8) aer partial sequence of the rat growth hormone according to Wallis und.Daviss _{s e} loc cit _{t /} comprising residues 1-43,

ta

29 # 8 _β 80 GZ 57 941 18

65-69, 108-113, 133-143 and 150-190, Table 3

DNA nucleotide sequence of a strand containing the entire rat growth hormone coding sequence. The corresponding amino acids, together with their positional number, are labeled with respect to the amino terminus corresponding to the sequence of pro-growth hormone. The corresponding mRNA sequence is same, only in the mRNA T is replaced by LJ;

29 * 8 _β 80 GZ 57 941 18

Table 3

•. Met Ala Ala

5 · -GTGGACAGATCACTGAGTGGCG ATG CCT CCA

Trp Pro Gin GIu Ala GIy Ala Leu Pro Ala Met Pro Leu Ser TGG CCT CM GAG CCT GGT GCT TTA CCT GCC ATG CCC TTC TCC

Ala Ala Asp Thr Tyr.Lye GIu Phe Glu Arg Ala, Tyr lie Pro CCT CCT CAC AGC TAC AAA GAG TTC GAG CGT CCC TAG ATT CCC

60 Ser Glu Thr He Pro Ala Pro Thr Gly Lys Glu Glu Ala Gin

TCA GAG ACC ATC CCA GCC CCC ACC GGC AAG GAG GAG CCC CAG

Ser Trp Leu Gly Pro VaI Gin Phe Leu Ser Arg He Phe Thr TCA TGG CTG CGG CCC CTG CAG TTT CTC AGC ATG ATC TTT ACC

120 AsP Leu Glu Glu Gly He Gin Ala Leu Met Gin Glu Leu Glu '

GAC CTG GAA CAC GCC ATC CAG CCT CTG ATG CAG GAG CTG GAA

Phe Asp Ala Asn Met Arg Ser Asp Asp Ala Leu Leu Lys Asn TTT GAC CCC AAC ATG CCC AGC GAT GAC GCT CTG CTC AAA AAC

ISO Tyr Leu Arg VaI Met Lys Cys Arg Arg Phe AIa Glu Ser Ser

TAG CTG CGG CTC ATG AAC TGT CGC CGC TTT CCG GAA ACC ACC CTGCCAACTGCCACCCCTACACTTTGTCCTAATAAAATTAATGATGCATCATATC

29 * 8. 80 GZ 57 941 13

(Continuation)

Asp Ser CIn Thr Pro Trp Leu Leu Thr Phe Ser Leu Leu Cys CAC TCT CAG ACT CCC TGG CTC CTG ACC TTC AGC CTG CTC TCC

'20

eu Leu Phe AIa Asn Ala VaI Leu Arg Ala Gin His Leu His ACT CUG TTT CCC AAT GCT GTG CTC CCA CCC CAG CTAC CAC

40.

GIu GIy Gin Arg Tyr Ser Gin Asn Ala Gin Ala Ala Phe CAG GGA CAC CGC TAT TCC ATT CAG AAT GCC CAG GCT GCT TTC

8o GIn Arg Thr Asp Met Glu Leu Leu Arg Phe Ser Leu Leu Leu CaG AgA ACT GAC ATG GAA TTG CTT CGC TTC TCG CTC CTG CTC

100 Ash Ser Leu Met Phe GIy Thr Ser Asp Arg VaI Tyr Glu Lye AAC AGC CTG ATG TTT GGT ACC TCG GAC CGC GTC TAT GAG AAA

140 Asp GIy Ser Pro Arg JIe 'GIy Gin He Leu Lys GIn Thr Tyr GAC GGCAGC CCC CGT ATT GGG CAG ATC GTC AAG CAA ACC TAT

160 Tyr GIy Leu Leu Ser Cys Phe Lys Lys Asp Leu Hie Lys AIa TAT GGG CTG CTC TCC TGC TTC AAG AAG GAC CTG CAC AAG CCA

Cyβ Ala Phe

TGT GCT TTC TAG GCACAGAGTGGTGTCTCTGCGGCACTCCCCCGTTACCGC

poly (A) - 3 '

Leu Leu CTG CTG

GIn Leu GAG CTG

Cys Phe TCC TTC

Hey GIn ATC CAG

Leu Lys CTG AAG

Asp Lys GAC AAG

GIu Thr GAG ACC

29 »8,

GZ 57 941

- 53 -

(Continuation)

CTGTACT

29 »8, 80 GZ 57 941 18

The isolation of human goitrogen hormone mRNA is essentially as described in Example 7, except that the biological source is human hypo-soympathetic tissue, five benign human pituitary tumors that have been frozen in liquid nitrogen after surgical removal, and each of which -0.4 to 1.5 g weighed '* were in 4molarer Gunidiniumthiocyanat solution which had been buffered at Imolar mercaptoethanol and adjusted to pH 5.0, thawed at 4 ⁰ C and homogenized * the homogenate was 1.2 ml 5, 7 molar cesium chloride solution containing 100 mmol of ethylenediaminetetraacetic acid was added and centrifuged for 18 hours at 37,000 revolutions / minute in rotor SW 50,1 of a Beckraann ultracentrifuge at 15 ° C. »The RNA was placed on the bottom of the centrifuge tube * Further purification with a column with oligo-dT and sucrose gradient sedimentation was carried out as in Examples 1, 2 and 3r about 10 % ö.er so isol Formed RNA-encoded growth hormone was estimated to be from the incorporation of a radioactive amino acid precursor into anti-growth hormone precipitate in a cell-free wheat cell translation cell; compare Roberts, B * E. and Patterson, B _e H, Proc «N-at _e Cecad * Be« USA / Ό, 2330 (1973) «. The production of cDM8 "from human growth hormone is essentially as described in Example 7." The cDNA is fractionated by gel electrophoresis and the material migrating to a position corresponding to about SOO nucleotides in length is selected for clearing. Polyraerase 1, as described in Example 6, 'then takes place end-addition of hindrance

29 * S * 80 GZ 57 941 18

ΣΙΪ-links "The cDNA is then recombined with the treated with alkaline phosphatase Plasrnid pBR-322 and DNA ligase The rekombiriierten DNS E ₀ coli.X-1776 is transformed, and a growth hormone DNA containing strain is selected" This strain is grown in preparative amounts, the DNA is isolated therefrom, and their Nucleotidseque'nz is determined _e / achstumshormon the cloned Sn ~ contains DNA encoding the human growth hormone coding nucleotides * are the entire amino acid sequence the first 23 amino acids of human growth hormone

Io HpN-P ^ e-Pr o ~ Thr-lie-Pro-Leu »Se r-Arg-Leu-Ph e» Asp-Asn-Ala-Met

20 ~ Leu-Arg-Ala ~ His-Arg-Leu-His-Leu-Gln ~ _e

The rest of the sequence shows the table 4%

Table 4 "".

Nucleotide sequence of a strand of human growth hormone DNA "The numbers refer to the amino acid sequence of the human growth hormone, starting at the Am'ino ~ end * The DNA sequence corresponds to the mRNA sequence for human growth hormone, replacing only the T in the fliRNA-U »'

29c S ₃ 80 GZ 57 941 18

Table 4

24 34

Ala Phe Asp Thr Tyr Gin GIu Phe GIu GIu Ala Tyr

G. GCC TTT GAC ACC DAY CAG GAG TTT GAA GAA GCC ACT

60 Thr Ser Leu Cys Phe Ser GIu Ser He Pro Thr per Ser ACC TCC CTC TGT TTC TCA GAG TCT ATT CCG ACA CCG TCC

80 Arg lie Ser Leu Leu Leu lie Gin Ser Trp Leu GIu Pro CGC ATC TCC CTG CTG CTC ATC CAG TCG TGG CTG GAG CCC

GIy Ala Ser Asp Ser Asn VaI Tyr Asp Leu Leu Lys Asp GGC GCC TCT GAC AGC AAC GTC TAT GAC CTC. CTA AAG GAC

140 GIy Ser · Pro Arg Thr Gly Gin He Phe Lys Gin Thr Tyr CCC AGC CCC CCG ACT GGG GAG ATC TTC AAG CAG ACC TAC

160 Lys Asn Tyr GIy Leu Leu Tyr Cys Phe Arg Lys Ar> p Met CAAG AAC TAC CCG CTG CTC TAC TGC TTC AGG AAG CAC ATG

191 Vai GIu Giy Ser Cys GIy Phe

CTG CAG GGC AGC TGT GGC TTC TAG CTCCCCGGGTGGCATCCCT

I?

29, 8, 80 GZ 57 941 18

- 57 -

Tabello 4 (continued)

He Pro Lys GIu Gin Lys Tyr Ser Phe Leu Gin Asn Pro Gin ATC CCA AAG CAA CAG MG TAT TCA TTC CTG CAG AEC CCC CAG

Asn Asg GLu Glu Thr Gin GIn Lys Ser Asn Leu Giu Leu Leu AAC AGG GAG CM ACA CAA CAG ΑΛΑ TCC AAC CTA. GAG CTG CTC

100

VaI Gin Phe Leu Arg Ser VaI Phe AIa Asn Asn Leu VaI ιyr GTG CAG TTC CTC AGG AGT CTC TTC GCC AAC AAC CTG CTG TAC

120 Leu GIu GIu Gly He Gin Thr Leu Met GIy Arg Leu Glu Asp CTA GAG CAA GCC ATC CAA ACG CTG ATG GGG AGG CTG GAA GAC

Ser Lys Phe Asp Thr Asn Ser His Asn His Asp Ala Leu Leu AGC AAG TTC GAC ACA AAC TCA CAC AAC CAT CAC TCA CTA CTC

180 Asp Lys VaI Glu Thr Phe Leu Arg He VaI Gin Cys Arg Ser GAC AAG GTC GAG ACA TTC CTG CGC ATC GTG CAG TCC CGC TCT

TGACCCCTCCCCACTGCCTCTCCTGGCC - ·

3 '

, £ £ 4 &<& έ, _{GZ 57 941 18}

By the method of the present invention, it becomes possible for the first time to isolate a nucleotide sequence coding for a specific regulatory protein of a higher organism, such as a vertebrate, further, the transfer of the genetic information contained therein can be carried out in a microorganism in which the unlimited replication The disclosed method can be applied to the isolation and purification of the human insulin gene and its transfer into a microorganism. "Likewise, human growth hormone gene and other protein genes can be isolated, transferred and replicated in a microorganism." Disclosed are some novel recombinant plasmids. which have in their nucleotide structure a structure obtained by transcription from a gene of a higher organism. "Disclosed are new microorganisms that are modified to contain a nucleotide sequence that is characterized by Transcription is originated from a gene of a higher organism "The examples illustrate the invention in the transfer of the rat gene for proinsulin I into a strain of Escherichia coli and in the transfer of the rat gene for growth hormone to a strain of E _f coli" The sequence of the main piece of the transferred gene was determined in each case, whereby it was found, as & they the entire amino acid sequence of rat proinsulin I or. " Rat growth hormone coded *

The microorganism described herein was deposited with the ATCC under accession number 3.1392. Furthermore, the rat insulin gene containing piasmide, which can be independently outbred to other microorganism strains, was deposited with the ATCC under the accession number 40003

Claims (1)

_Λ - « ^{29e 8β 80} 2k4 Z6 ?. , · GZ 57 941 18 , - 59 - A method for producing a DNA transfer vector with a growth hormone encoding nucleotide sequence, characterized in that (a) pituitary cells containing growth hormone-encoding nRNA isolated, (b) the mRNA from the pituitary cells extracted in the presence of an RNase inhibitor, whereby RNase degradation of the mRNA is prevented, (c) isolating substantially free mRNA from protein, DNA and other RNAs, (d) synthesizing a double-stranded cDNA having one strand of one of the mRNA has complementary nucleotide sequence., to obtain a cDNA having a nucleotide sequence encoding growth hormone, (e) providing a DNA transfer vector which has reactive ends capable of binding with each other or with double-stranded cDNA, and (f) connecting the DNA transfer vector with the double-stranded cDNA with a nucleotide sequence encoding growth hormone. 2 _e The method of item l _f characterized in that one uses a RNase inhibitor, which contains a chaotropic cation, a chaotropic anion and a disulfide cleaving agent ". 29o 8 «80 GZ 57 941 13 3 «Method according to item 2, characterized in that guanidinium ion is used as chaotropic cation and thiocyanation as chaotropic anion» '4 * method according to item 2 _f, characterized in that one uses ice Diaulfidbindungen-splitting agent ß-mercaptoethanol. Os method according to item 1 ₍ characterized by using as an RNase inhibitor 4-molar guanidinium thiocyanate and 0.2-molar ß-mercaptoethanol 6 "method according to item 1, characterized in that the reactive ends-owning DNA molecules - are linked in step (f) by a method in which a certain part of the reactive ends at the Ver. .knüpfung is prevented from each other _t the method is that treating a specific part of the reactive ends with a reagent capable of removing the 5 '~ terminal phosphate groups, and the DNA molecules having pretreated and unbehan ™ punched reactive terminals, incubated together with a DNA ligase which catalyzes a link between the end-producing reaction, with the exception of the pretreated ends f which are not linked together in the ligase-catalyzed reaction, 7 "Process according to item 6, characterized in that. For the pretreatment of the reactive ends of alkaline. Phosphatase used «. , ' 29 «8. 80 GZ. 57 941 18 An ancestor according to item 6, characterized in that the DNA molecules to be joined consist of a mixture of restriction-endonuclease-treated plasmid DNA and non-plasmid linear DNA and the part intended for treatment contains the plasmid DN3, so that the end-sn-end connection of the DNA without recombinant xon is prevented with the non-plasmid DNA "