DE2858357C2 - - Google Patents

Description

The application relates to the DNA indicated in claims 1 and 2 Transfer vectors. The claim 3 relates to a changed Microorganism containing the vector according to Claim 1.

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

DNS = Deoxyribonucleic acid RNS = Ribonucleic acid cDNA = complementary DNA (enzymatically synthesized from a mRNA sequence) mRNA = messenger RNA tRNA = transfer RNA dATP = Deoxyadenosine triphosphate dGTP = Deoxyguanosine triphosphate dCTP = Deoxycytidine triphosphate A = Adenine T = Thymine G = Guanine C = Cytosine Tris = 2-amino-2-hydroxyethyl-1,3-propanediol EDTA = Ethylenediamine tetraacetic acid ATP = Adenosine triphosphate TTP = Thymidine triphosphate

The biological significance of the base sequence of DNA is the a store of genetic information. It is known, that the base sequence of the DNS is used as code that the amino acid sequence of all those produced by the cell Proteins determined. In addition, parts of the sequence serve regulatory Purposes, for controlling production time and amount of each protein. The operation of this controlling Elements is only partially recognized. Finally, the Base sequence in each strand as a template in replication the DNA that accompanies cell division.

The way in which the information of the base frequency the DNA transferred to the amino acid sequence of proteins is a fundamental process that is universal for all is living organisms.  

It could be proven that each is usually in Proteins existing amino acid through the sequence of a or more trinucleotides or triplets. Each protein thus corresponds to a segment of DNA with a triplet sequence corresponding to the amino acid sequence of the Protein corresponds. Illustrates the genetic code following table.

Genetic code Phenylalanine (Phe) TTK Leucine (Leu) XTY Isoleucine (Ile) ATM Methionine (Met) ATG Valin (Val) GTL Serine (Ser) ORS Proline (Pro) CCL Threonine (Thr) ACL Alanine (Ala) GCL Tyrosine (Tyr) TAK termination TAJ termination TGA Histidine (His) CAK Glutamine (Gln) CAJ Asparagine AAK Lysine (Lys) AAJ Aspartic acid (Asp) GAK Glutamic acid (glu) GAJ Cysteine (Cys) TGK Tryptophan (Try) TGG Arginine (Arg) WGZ Glycine (Gly) GGL

Key: Each triplet of three letters represents a trinucleotide of DNA, with a 5 'left end and one 3'-end right. The letters stand for the purine or Pyrimidine bases that form the nucleotide sequence:

A = Adenine G = Guanine C = Cytosine T = Thymine X = T or C if Y = A or G X = C, if Y = C or T Y = A, G, C or T, if X = C Y = A or G if X = T W = C or A, if Z = A or G W = C, if Z = C or T Z = A, G, C or T if W = C Z = A or G if W = A QR = TC if S = A, G, C or T QR = AG, if S = T or C S = A, G, C or T if QR = TC S = T or C if QR = AG J = A or G K = T or C L = A, T, C or G M = A, C or T.

In the biological process of translation of the nucleotide sequence in an amino acid sequence is carried out as the first stage, the so-called Trancription. At this stage, a segment of the DNA with the sequence responsible for the protein to be produced transferred to an RNA. The RNA is similar to the DNA Polynucleotide in which, however, ribose instead of deoxyribose and uracil instead of thymine. The bases of the RNA are capable of the same type of base pairing as the bases the DNS. By transcription of a DNA nucleotide sequence resulting RNA is therefore complementary to this copied sequence. This RNA is called messenger RNA (mRNA) because of their intermediate position between the genetic apparatus and the apparatus of protein synthesis of the cell.

In the cell, the mRNA serves as a template in a complex Process involving numerous enzymes and organelles within  the cell are involved and the more specific to the synthesis Amino acid sequences leads. This process is called translation designated.

Often there are other stages that serve to get out of the during translation resulting amino acid sequence to produce functional protein. An example is the Production of insulin.

The direct precursor of insulin is a polypeptide, Proinsulin, which is the two insulin chains A and B contains bound via another peptide C, cf. Steiner, D.F., Cunningham, D., Spigelman, L. and Aten, B., Science 157, 697 (1967). Recently it was reported that the first translation product of insulin mRNA is not Proinsulin itself, but a pre-proinsulin is that more than 20 additional amino acids at the amino end of proinsulin contains, cf. Cahn, S.J., Keim, P. and Steiner, D.F., Proc. Natl. Acad. Sci. USA 73, 1964 (1976), and Lomedico, P.T. and Saunders, G.F., Nucl. Acids Res. 3, 381 (1976). The structure of the pre-proinsulin molecule can schematically represented as follows: NH₂- (Pre-peptide) - B-chain- (C peptide) -A chain-COOH.

Numerous proteins of importance for medicine or research become in the cells of higher organisms like the vertebrates found or produced. These include, for example, the Hormone insulin, other peptide hormones like growth hormone, proteins involved in the regulation of blood pressure and numerous enzymes with relevance to technical, medical or research purposes. It is often difficult to get these proteins in useful quantities by extraction from the organism to gain, especially in human proteins  Origin. There is therefore a need for techniques through such proteins from cells outside the organism be generated in reasonable quantities. In some cases It is possible to use suitable cell strains through the techniques to preserve the tissue culture. The growth of cells in However, tissue culture is slow, the medium is expensive, the conditions must be carefully controlled and the Yields are low. Often it is also difficult a cultured cell strain with the desired differentiated To get properties.

In contrast, microorganisms like bacteria relatively easily bred in chemically defined media become. The fermentation technology is already highly developed and easy to control. The growth of organisms is fast, and high yields are possible. also Certain microorganisms were already genetically accurate characterized and in fact among the best characterized and recognized organisms.

It would therefore be very desirable to transfer a gene code for a protein of medical importance from an organism, which usually produces the protein in one to achieve a suitable microorganism. In this way The protein could be under controlled growth conditions produced by the microorganism and in desired amounts to be obtained. It is also possible that the total cost the production of the desired protein by such a Procedure could be significantly reduced. The ability for isolating and transferring the gene sequence containing the Production of a special protein destined to one Microorganism of precisely known genetic structure opened besides, the research the valuable possibility to study how the synthesis of such a protein  controlled and how the protein is processed after synthesis becomes. Furthermore, isolated genes can be altered so that They protein variants with other therapeutic or functional Code properties.

The present invention relates to measures to the mentioned To achieve goals. A method is disclosed the multiple stages, including enzyme-catalyzed Reactions include. Below are the previous knowledge outlined on the nature of these enzyme reactions.

Revese transcriptase catalyzes the synthesis of one RNA template complementary DNA in the presence of the RNA template, an oligo-deoxynucleotide primer and the four deoxynucleoside triphosphate dATP, dGTP, dCTP and TTP. The reaction will initiated by the noncovalent binding of the oligo- deoxynucleotide primer to the 3 'end of the mRNA, on top follows the stepwise addition of the appropriate deoxynucleotides, according to the base pairing rule, to the 3 'end the growing chain. The product obtained as a product can be described as hairpin shaped, it contains the parent RNA and a complementary DNA single strand. The reverse transcriptase may also have a similar reaction catalyze a DNA single strand as a template, in which case the product is a hairpin-shaped DNA Double strand receives DNA single strand with a loop, through which a pair of ends are connected; see. Aviv, H. and Leder, P., Proc. Natl. Acad. Sci. USA 69, 1408 (1972), and Efstratiadis, A., Kafatos, F.C., Maxam, A.F. and Maniatis, T., Cell. 7, 279 (1976).  

Restriction endonucleases are enzymes responsible for hydrolysis of phosphodiester bonds in double-stranded DNA are, wherein the sequence of the DNA strand is cleaved. If the DNA is in the form of a closed loop, so this is converted into linear form. The main feature an enzyme of this kind is that the hydrolytic Effect occurs only where a specific nucleotide sequence is present. This sequence is called a recognition site (Cleavage site) of the restriction endonuclease. Restriction endonucleases have been selected from several Sources isolated, and their nucleotide sequence and the recognition sites were determined. Some restriction endonucleases hydrolyze the phosphodiester bonds in both strands the same place, so that blunt ends arise. Other catalyze the hydrolysis of bonds by some Nucleotides are separated from each other, creating single-stranded ones Regions arise at each end of the cleaved molecule. These single-stranded ends are self-complementary, therefore Cohesive, and they can be used to hydrolyzed Bind DNA again. Because each for a split by one such enzyme prone DNA the same recognition site the same cohesive ends are produced, so that it becomes possible to have heterologous DNA sequences to link with other, similarly obtained sequences; see. Roberts, R.J., Crit. Rev. Biochem. 4, 123 (1976). The recognition sites are relatively rare, but the general utility of restriction endonucleases greatly expanded by the chemical synthesis of double-stranded Oligo-nucleotides with the sequences of the recognition sites. Thus, virtually every DNS segment can be transferred to another segment be coupled by simply selecting the appropriate restriction oligo-nucleotide attaches to the molecular ends and that Product of the hydrolytic action of the corresponding restrictive Subjecting endonuclease, taking the required  receives cohesive ends; see. Heynecker, H.L., Shine, J., Goodman, H.M., Boyer, H.W., Rosenberg, J., Dickerson, R.E., Narang, S.A., Itakura, K., Lin, S. and Riggs, A.D., Nature 263, 748 (1976), and Scheller, R.H., Dickerson, R.E., Boyer, H.W., Riggs, A.D. and Itakura, K., Science 196, 177 (1977).

S1 endonuclease is an enzyme used to hydrolyze the phosphodiester bonds single-stranded DNA or single-stranded Intermediate pieces in otherwise double-stranded DNS is capable; see. Vogt, V.M., Eur. J. Biochem. 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 causes the Beispsiel are formed by two DNA fragments that are cohesive Ends are held together. The normal function of the Enzyme probably consists of single-stranded intermediates in an otherwise double-stranded DNA molecule. Under appropriate conditions, however, DNA ligase can be used Catalyst blunt ends where two molecules catalyze covalently bound with blunt ends; see. Sgaramella, V., Van de Sande, J.H., and Khorana, H.G., Proc. Natl. Acad. Sci., USA 67, 1468 (1970).

Alkaline phosphatase is an enzyme, the phosphate ester bonds 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. As plasmid one calls every autonomous DNA-replicating unit in a microbial cell may be present next to the genome of the host cell. On Plasmid is not genetic with the chromosomes of the host cell  connected. Plasmid deoxyribonucleic acids are double-stranded Ring molecules, generally with molecular weights on the order of a few million, though in some the molecular weight is greater than 10⁸. you usually represent only a small percentage the entire DNA of the cell. The plasmid DNA can be generally from the DNA of the host cell due to the separate large size difference. The plasmids can be independent of the speed of division the host cell can replicate, and in some cases their replication speed by changing the Growth conditions are influenced by the experimenter. Although the plasmid is a closed ring, can you artificially insert a DNA segment into the plasmid introduce a recombinant plasmid with enlarged Molecular size arises without losing its ability to Replication or expression of any genes of the plasmid would be significantly impaired. The plasmid therefore serves as a useful vector for transferring a DNA segment into a new host cell. Suitable for recombination of DNA Plasmids typically contain genes that are out Selection reasons may be useful, for example, genes drug resistance.

The apparent method may be understood by one of ordinary skill in the art readily on the isolation of a gene from transmitted to other organisms, including humans become.  

The ability to produce a DNA with a specific Sequence that defines the genetic code for a specific Release protein allows modification of the nucleotide sequence by chemical or biological means such, that also the finally produced protein is modified. For example, this could be a modified one Make growth hormone based on special medical Needs is aligned. The genetic ability to Production of a growth hormone-like amino acid sequence with the essential functional properties of the growth hormone can therefore affect a microorganism be transmitted.

The ability to transfer the genetic code for a certain protein that in the normal metabolism of a higher organism is needed in a microorganism such as a bacterium, opens up new possibilities for the production of such proteins. This also arises the possibility of propagating or replacing such Proteins by proteins, which changed from according to the invention Microorganisms were produced where the Ability of higher organism to normal production of these proteins was damaged.

The invention relates to a method for the isolation of a specific nucleotide sequence containing genetic information, the synthesis of DNA with this specific Nucleotide sequence and the transfer of the DNA into a host Microorganism.  

The invention is illustrated by specific DNS Sequences that are transferred to a bacterium, viz of the gene for human Growth hormone. In the present process is a selected cell population first for a new one Isolated method. The cells become intact mRNAs extracted a method in 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 for synthesis of a complementary (cDNA) strand required four Deoxynucleoside triphosphate occurs. The product of this first reaction with reverse transcriptase is in one Process further treated the ribonucleotide sequence selectively removed. The remaining deoxynucleotide sequence, which is complementary to the starting mRNA is expressed in a second reaction with reverse transcriptase or DNA polymerase in the presence of the four deoxynucleoside triphosphates incubated. The resulting product is one double cDNA structure, their complementary strands one end by a single-stranded loop are connected. This product is then used for the single strand specific nuclease treats the single-stranded one Split off loop. The resulting double-stranded cDNA is then lengthened by placing a adds specific DNA that represents the sequence of a recognition or cleavage site of a restriction enzyme. The Addition is catalyzed by a DNA ligase. The extended one cDNA is then probed with a restriction endonuclease treated at the 5 'ends of each strand of the double helix self-complementary single-stranded ends arise.  

A plasmid DNA with a recognition site for the same Restriction endonuclease is treated with this enzyme to cleave the polynucleotide strand and self-complementary to generate single-stranded nucleotide sequences at the 5 'ends. The 5'-terminal phosphate groups on the single-stranded Ends are removed to prevent the plasmid a ring structure capable of transforming a host cell forms. The cDNA prepared as described above and the plasmid DNA are then in the presence of DNA ligase incubated together. Under the reaction conditions described can be the formation of a viable, closed Ringes made from plasmid DNA only if a segment of the cDNS is inserted. The plasmid containing the cDNA sequence is then transferred to a suitable host cell. cells, who have taken a viable plasmid recognizes the appearance of colonies with a plasmid contributed Characteristic such as drug resistance. Pure bacterial strains containing the recombinant plasmid containing the built-in cDNA sequence are then grown, and the recombined plasmid is subsequently reisolated. In this way, large amounts of recombined Plasmid DNA are produced, from which one the specific cDNA sequence by endonucleolytic cleavage isolate again with the appropriate restriction enzyme can.  

The present invention comprises a process for isolation a DNA molecule with a specific nucleotide sequence and their transfer into a microorganism, wherein the original nucleotide sequence of the DNA after multiplication found in the microorganism.

The steps constituting the process according to the invention can be divided into four general categories:

1. The isolation of a desired cell population from a higher organism

There are two sources for a genetic sequence, the one special protein encoded: the DNA of the underlying Organism itself and a RNA translation of the DNA. To the current safety regulations of the National Institutes of Health is prescribed for the US that human Genes of any kind only in recombined DNA and then can be introduced into bacteria after being very carefully have been cleaned or if you are in specially equipped (P4) facilities works, cf. US Federal Register, Vol. 41, No. 131, 07. 07. 1967, pp. 27902-27943. each Process with actual usability for production of human protein, as for example the present  Method, therefore, preferably works with the insulation the specific mRNA having a nucleotide sequence, which encodes the desired protein. Enjoy this way one has the further advantage that the mRNA is lighter as DNA extracted from cells can be purified. In particular, one can also take advantage of the fact that in highly differentiated organisms such as the vertebrates, the identification of a specific cell population of special localization in the organism possible whose function is mainly in the production of the protein in question. Such a population can also be during a temporary stage of development of the organism. In such cell populations has a large proportion of those isolated from the cells mRNA is the desired nucleotide sequence. The choice of too isolating cell population and that during isolation Applied methods can be used in terms of output purity the mRNA is essential.

In most tissues, glands and organs become the cells from a generally fibrous network of connective tissue held together, which mainly consists of collagen, however, depending on other structural proteins, polysaccharides and mineral deposits. The isolation of cells of a particular tissue requires inevitably procedures to release the cells from the Connective tissue matrix. The isolation and cleaning of a special differentiated cell type therefore comprises two main stages: the secretion of cells from connective tissue, and the Separation of the desired cells from all others in the Tissue present cell types. The inventively provided Working methods are numerous on the isolation Cell types of different tissues applicable.  

Often, the proportion of desired mRNA can be increased if correct the cellular reactions to stimuli from the environment starts. For example, treatment with a Hormone to increase production of the desired mRNA to lead. Other methods are breeding at a particular Temperature or in the presence of a specific Nutrient medium or other chemical substance.

2. Extraction of the mRNA

An essential feature of the present invention is the virtually complete removal of RNase activity in Cell extract. The mRNA to be extracted is a single-stranded one Polynucleotide, unpaired with a complementary strand. The hydrolytic splitting of a single phosphodiester bond in the sequence, therefore, the entire molecule would be responsible for  the purpose of transferring an intact genetic Render the sequence unusable in a microorganism. As already mentioned, the enzyme RNase is widespread, active and exceptionally stable. It turns up the skin survives the usual rinsing procedures for glassware and occasionally contaminates stocks organic chemicals. The difficulties are special acute when working with extracts of pancreatic cells, because the thyroid gland is a source of digestive enzymes and therefore rich in RNase. The problem However, RNase contamination is present in all Tissues, and the method disclosed herein for removal of RNase activity is on all tissues applicable.

When the cells are ruptured and during all processes, for the isolation of protein virtually free RNA are applied, according to the invention is a combination from a chaotropic anion, a chaotropic cation and a disulfide bond-splitting agent.

The choice of suitable chaotropic ions is based on their Solubility in aqueous media and their availability. Suitable chaotropic cations are, for example, guanidinium, Carbamoylguanidinium, guanylguanidinium, lithium ion and like. Suitable chaotropic anions are, for example iodide, perchlorate, thiocyanate, diiodo-salicylate and  like. The effectiveness of salts by combination of such cations and anions is formed partly from their solubility. Such is for example Lithium diiodo salicylate a stronger denaturant as guanidinium thiocyanate, but it has a solubility only about 0.1M, and it is relatively expensive. The preferred combination of cation and anion is in the Guanidinium thiocyanate, as this salt is readily available and is readily soluble in aqueous media, up to about 5 molar Solutions.

Thiol compounds such as the β- mercaptoethanol are known to cleave intramolecular disulfide bonds in proteins by means of a thiol-disulfide exchange reaction. Numerous thiol compounds are effective in this direction, for example, β- mercaptoethanol, dithiothreitol, cysteine, propanol dimercaptan, and the like. An essential property is the solubility in water, since the thiol compounds in large excess on the intramolecular disulfides must be present to complete the exchange reaction. β- mercaptoethanol is preferred because of its availability and its low price.

In the inhibition of RNase during RNA extraction from cells or tissues is the action of a chaotropic salt directly depending on his concentration. The preferred concentration is therefore the highest possible concentration. The success of inventive method with respect to conservation intact mRNA during extraction is believed to be due the rapid denaturation of RNase and the extent of denaturation. This may explain the superiority of Guanidinium thiocyanate towards the hydrochloride, despite the fact that the hydrochloride is used as a denaturant only slightly weaker. The effectiveness of a denaturant is defined as the threshold concentration used for  complete denaturation of a protein is needed. On the other hand, the speed of denaturation depends numerous proteins from the concentration of the denaturant with regard to the threshold concentration, increased from the fifth to the tenth power; see. Tanford, C.A., Adv. Prot. Chem. 23, 121 (1968). This relationship gives qualitatively, that only slightly stronger Denaturant as guanidinium hydrochloride Protein at the same concentration many times over can denature faster. The kinetic relationships between RNase denaturation and mRNA preservation during their extraction from the cells was at the time of the invention apparently not recognized yet. The above considerations lead to the fact that the preferred denaturant a Such would be a low threshold concentration in Combination with high water solubility possesses. For this Reason is guanidinium thiocyanate over Lithiumdiiodsalylat although the latter compound is stronger Denaturant is because the solubility of guanidinium thiocyanate is much higher and therefore it is in one Concentration may be applied, allowing for faster RNase inactivation allowed. The above considerations also explain the preference of guanidinium thiocyanate before the comparatively soluble Hydrochloride, the former being a slightly stronger denaturant is.

The use of a disulfide bond-cleaving agent in combination with a denaturant enhances and enhances the effect of the latter, thus fully unfolding the RNase molecule. The thiol compound accelerates the progress of the denaturation process by preventing rapid renaturation, which may occur if the intramolecular disulfide bonds remain intact. Furthermore, any RNase remaining and contaminating the mRNA preparation becomes virtually inactive, even in the absence of denaturant and thiol. Thiol group disulfide bond-cleaving agents are effective to some extent at any concentration, but generally, a large excess of thiol groups over the intramolecular disulfide bonds is preferred for the equilibrium reaction to proceed in the direction of disulfide cleavage. On the other hand, many thiol compounds are malodorous and unpleasant to process at high concentrations, so that in practice arises an upper concentration limit. For the b- mercaptoethanol, concentrations in the range of 0.05M to 1.0M were found to be effective.

The pH of the medium during mRNA extraction from the Cells can range from pH 5.0 to 8.0.

After rupturing the cells, the RNA is removed from the mass separated from cell protein and DNA. For this purpose are Several methods are known, all of which are suitable. A common practice is a precipitation process with ethanol, where the RNA precipitates selectively. Preference is given to the invention bypassed the precipitation stage and the homogenate directly to a 5.7molare cesium chloride solution, which in a Centrifuge tube is located, piled, followed by centrifugation he follows; see. the description of glisine, V., Crkvenjakov, R., and Byus, C., Biochemistry 13, 2633 (1974). This method is preferred because it is consistently one for RNase maintains unfavorable environment, so that the RNA in high yield and free of DNA and protein gains.  

The above procedures result in purification of the entire RNA from the cell homogenate. However, only part of this RNA is the desired mRNA. To further purify this mRNA, used one the fact that in the cells of higher organisms mRNA after transcription in the cell further processed becomes under binding with polyadenylic acid. Such mRNA with attached poly A sequences can be selectively isolated are chromatographed on columns of cellulose, to which oligo-thymidylate is attached, see Aviv, H., and Leather, P., loc. cit. The above processes provide in essential pure, intact mRNAs from starting materials, that are rich in RNase. The cleaning of the mRNA and the following in vitro steps can be performed with each mRNA, regardless of its source organism, in practical be carried out in the same way.

Under certain conditions, for example when using of cells from tissue culture as a source of mRNA, the contamination with RNase may be so low that above stage of RNase inhibition is not required. In these cases, the methods of the prior art sufficient to reduce the RNase activity.

3. Formation of the cDNA

Fig. 1 is a schematic representation of the remaining stages of the process. 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 transcriptase, although in principle any enzyme capable of forming a complementary strand of DNA due to the mRNA used as the template could be used. The reaction can be carried out under the known conditions, using the mRNA as template and a mixture of the four deoxynucleoside triphosphates as precursors of the DNA strand. Suitably, one of the deoxynucleoside triphosphates in α - position with 32 _p marked to follow the course of the reaction and the workup and separation, for example by chromatography and electrophoresis, to control and make quantitative yield data can; see. Efstratiadis, A., et al., Loc. cit. As can be seen from the drawing, the product of the reaction with the reverse transcriptase is a double-stranded hairpin with noncovalent binding between the RNA strand and the DNA strand.

The product of the reaction with the reverse transcriptase is recovered from the reaction mixture by standard procedures. Appropriately, one uses a combination of Phenol extraction, Chromatography on "Sephadex G-100" and precipitation with ethanol.

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

After the alkaline hydrolysis and subsequent neutralization of the 32 _p-labeled cDNA may be concentrated by precipitation with ethanol, if desired.

The synthesis of a double-stranded hairpin-shaped cDNA is done using the appropriate enzyme, for Example with DNA polymerase or reverse transcriptase.  

The reaction conditions are similar to that described above, including the use of a labeled in a position with 32 _P nucleoside triphosphate. Reverse transcriptase is available from a variety of sources, for example from avian myeloblastosis virus (the virus is available from Life Sciences Incorporated, St. Petersburg, Florida, which produces it under a contract with the National Institutes of Health).

After the formation of the cDNA hairpin it may be recommended be to extract the DNA from the reaction mixture purely. Again, you can conveniently phenol extraction, chromatography on "Sephatex G-100" and precipitation with ethanol for purification of the DNA product and liberation of pollutants Use protein.

The hairpin structure can be transformed into a common double-stranded one DNA can be transferred by using the single-stranded loop, which connects the ends of complementary strands removed. Various enzymes are available for this purpose the one specific hydrolytic cleavage single-stranded Cause DNS areas. One suitable for this purpose Enzyme is the S1 nuclease from Aspergillus oryzae (the Enzyme may be obtained from Miles Research Products, Elkhart, Indiana, be obtained). The treatment of hairpin DNA with S1 nuclease leads to high yields of cDNA molecules with base-paired ends. Extraction, Chromatography and ethanol precipitation as described above. The use of reverse transcriptase and S1 nuclease in syntheses of double-stranded cDNA transmissions of mRNA was reported by Efstratiadis et al., loc. cit.  

Optionally, the proportion of cDNA molecules with blunt ends increase by treatment with E. coli DNA Polymerase I in the presence of the four deoxynucleoside triphosphates. The combination of exonuclease and polymerase Activity of the enzyme leads to the removal of all protruding 3 'ends and to fill up all protruding 5 'ends. In this way, the participation of the Major amount of cDNA molecules in the following linkage reactions ensured.

The next stage of the procedure is the treatment of Ends of the cDNA product such that appropriate sequences standing at each end, which is a recognition point for one Restriction endonuclease have. The choice of the to Ends to be added DNA fragment is determined by Practical reasons in the handling. The at the ends sequence to be added will be selected according to the selected Restriction endonuclease selected, and this choice is based turn on the choice of the DNA vector with which the cDNA to recombine. The chosen plasmid should be at least own a post that is due to the split Restriction endonuclease responsive. The plasmid pMB9 has, for example, a recognition site for the enzyme Hind III. Hind III is isolated from Hemophilus influenza and by the method of Smith, H.O., and Wilcox, K.W., J. Mol. Biol. 51, 379 (1970). The enzyme Hae III out Hemophilus aegyptious is produced by the method of Middleton, J.H., Edgell, M.H., and Hutchinson III, C.A., J. Virol. 10, 42 (1972), purified. An enzyme from Hemophilus suis known as Hsu I, catalyzes the hydrolysis to the Same recognition sites as Hind III. These two enzymes are therefore considered to be functionally interchangeable.  

Conveniently, a chemically synthesized double-stranded decanucleotide having the recognition sequence for Hind III is used to bind to the ends of the cDNA double strand. The double-stranded decanucleotide has the sequence shown in FIG. 1, cf. Heyneker, HL, et al., And Scheller, RH, et al., Loc. cit. Several such synthetic recognition site sequences are available today so that the ends of the double DNA can be prepared to respond to the action of one of the numerous restriction endonucleases.

The binding of the recognition site sequence to the ends of the cDNA can be done in a known way. The method of choice is a reaction called "blunt-end linkage" which is catalyzed by a DNA ligase purified by the method of Panet, A., et al., Biochemistry 12, 5045 (1973) , The blunt-end junction reaction was reported by Sgaramella, V., et al., Loc. cit., described. The product of linking a blunt-ended cDNA to a large molar excess of a double-stranded decanucleotide having a Hind III endonuclease recognition site is a cDNA with this Hind III recognition sequence at 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 'ends, see FIG. 1.

4. Formation of a recombinant DNA transporter vector

In principle, a variety of viral and plasmid DNA can be used to recombine with the cDNA prepared as described above. The basic prerequisites are that the DNA transfer vector must be capable of entering a host cell, replicated in the host cell, and also having a genetic determinant that allows for the outgrowth of those host cells that have taken up the vector , For reasons of public safety, the range of selection should be limited to those transfer vector species that appear appropriate for the particular experiment, in accordance with the aforementioned guidelines of the National Institutes of Health. The list of approved DNA transfer vectors is constantly expanding as new vectors have been developed and approved by the National Institutes of Health's equivalent committee. Of course, the invention contemplates the use of any virus and plasmid DNA having the described capabilities, including those whose approval is yet to come. Suitable transfer vectors, the use of which is approved today, are various derivatives of bacteriophage λ (cf., for example, Blattner, FR, Williams, BG, Blechl, AE, Denniston-Thompson, K., Faber, HE, Furlong, LA, Grunwald , DJ, Kiefer, DO, Moore, DD, Schumm, JW, Sheldon, EL, and Smithies, O., Science 196, 161 [1977]) and derivatives of the plasmid col El (see, for example, Rodriguez, RL, Bolivar, S., Goodman, HM, Boyer, HW, 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. 471-477). Plasmids from col E1 are relatively small, have molecular weights on the order of less than millions, and have the property that the number of copies of plasmid DNA per host cell can be increased from 20 to 40 under normal conditions to 1000 or more when using the host cell treated with chloramphenicol. The ability to increase the amount of gene in the host cell, under certain circumstances, allows the host cell under the control of the experimenter to produce mainly proteins encoded by the genes on the plasmid. Such derivatives of col El are therefore preferred transfer vectors according to the invention. Suitable derivatives of col El include plasmids pMB-9 bearing the gene for tetracycline resistance, and pBR-313, pBR-315, pBR-316, pBR-317, and pBR-322 which contain a gene other than the tetracycline resistance gene for ampicillin resistance. 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. In the examples of this specification, a plasmid was always used from col El, which in addition to the drug resistance gene possessed a Hind III recognition site.

As with the plasmid, may also be the choice of the appropriate host taken from a very broad spectrum, the area However, for reasons of public security, close was limited. An E. coli strain called X-1776 was developed and approved by the National Institutes of Health approved for methods of the type described, P2 devices are required; see. Curtiss III, R., Ann. Rev. Microbiol. 30, 507 (1976). E. coli RR-1 can be used when there are P3 facilities. As in the case of plasmids, the invention will be understood the use of strains of any host cells that can serve to accommodate the chosen vector, including Protists and other bacteria, for example Yeasts.  

Recombinant plasmids are obtained by mixing with Restriction endonuclease treated with plasmid DNA cDNS mixed, both analog end groups contain. To the possibility that cDNA segments combinations to go into each other, to keep it minimal the plasmid DNA was used in molar excess via the cDNA. This way of working has so far that the bulk of the plasmids without an inserted cDNA fragment circulates. The subsequently transformed Cells then contain mainly plasmid and no plasmids recombined with cDNA. The result is a very lengthy and time-consuming selection process. According to the prior art, this problem has been solved by one tried to make DNA vectors with a recognition site for the restriction endonuclease in the middle of a suitable labeling gene such that the insertion of the cDNA shares the gene, resulting in loss of the gene encoded by the gene Function occurs.

Preferably, a method of decreasing colony counts to be examined for recombined plasmids is used. Here, plasmid DNA cut with the restriction endonuclease is treated with alkaline phosphatase (commercial enzyme). By treating with alkaline phosphatase, the 5'-terminal phosphate groups are removed from the endonuclease-generated ends of the plasmid, and self-assembly of the plasmid DNA is prevented. The ring formation and transformation thus depend on the insertion of a DNA fragment having 5'-phosphoylated ends. This method reduces the relative frequency of transformations without recombination to less than 1 to 10 ⁺⁴ .

The method according to the invention is based on the fact that the reaction catalyzed by DNA ligase takes place between a 5'-phosphate-DNA end group and a 3'-hydroxyl end group DNS. Becomes the terminal 5'-phosphate group removed, so no binding reaction occurs. Is to connect double-stranded DNS, so are two situations possible, as shown in Table 1:

Table 1

In Table 1, the double-stranded DNA is schematically indicated reproduced two parallel lines, with the 5 'and 3' End groups depending on hydroxyl or phosphate groups. In case I, 5'-phosphate groups are present on both reactive ones End groups, with the result that both Strands are covalently bound. In case II owns only one of the strands to be connected has a terminal 5'-phosphate group with the result that a single-stranded covalent  Bonding occurs while in the other strand a discontinuity is present. The non-covalently linked strand remains with the molecule due to hydrogen bonding between complementary base pairs to the opposite Strands connected in a known manner. In case III owns none of the ends of the reactants has a 5'- Phosphate group, so that no binding reaction occur can.

Unwanted binding reactions can therefore be prevented by adding appropriate ends of the reactants, whose connection is to be prevented treated under Removal of the 5'-phosphate groups. You can do any method for the removal of 5 'phosphate groups that the DNS not otherwise damaged. Preferably, the hydrolysis catalyzed by the enzyme alkaline phosphatase.

The process described above is also useful, if a linear DNA molecule is divided into two subfragments, typically with a restriction endonuclease, too cleave and then reconstruct in the original sequence is. The subfragments can be specially cleaned and the desired sequence can be reconnected the subfragments are reconstituted. The enzyme DNA Ligase, which is the end-to-end linkage of DNA fragments catalyzed, can be used for this purpose, cf. Sgaramella, V., Van de Sande, J.H., and Khorana, H.G., Proc. Natl. Sci. USA 67, 1468 (1970). Are the to be connected Sequences not blunt, so you can get those from E. coli Use ligase, cf. Modrich. P., and Lehman, I.R., J. Biol. Chem. 245, 3626 (1970).  

The reconstitution of the original sequence by restriction Endonuclease obtained subfragments becomes strong improved, if one uses a method, the reconstitution prevented in the wrong sequence. this happens by treating the cDNA fragment of homogeneous length desired sequence with an agent containing the 5'-terminal Phosphate groups of the cDNA removed before cleavage of the homogeneous cDNA with a restriction endonuclease. you again preferably uses alkaline phosphatase. The 5'-terminal phosphate groups are a structural prerequisite for the subsequent binding effect of DNA ligase, the reconstitution of the split subfragments is used. Ends without 5'-terminal phosphate group therefore can not be bound covalently. The DNA subfragments can only be bound to those ends that have a 5'-phosphate group.

This procedure prevents the most important unwanted Binding reaction, namely the joining of two fragments with reverse sequence, ie back-to-front instead front-to-rear. Other possible side reactions, such as dimerization and cyclization are not prevented as these in a Type II reaction according to Table 1. These Side reactions are less detrimental as they are too physical separable and identifiable products lead what to the new combination in the wrong direction is not the case.  

The described method is generally applicable to the Isolation and purification of a gene from a higher organism, including human genes, its transfer into a microorganism and replication in the microorganism. Also, new recombinant plasmids containing all or one Part of the isolated gene are described. Further previously unknown new microorganisms are described, the as part of their genetic makeup a gene of a higher one Contain organism. The example also recombinant plasmids characterizes the parts of human  Contain growth hormone gene and the genes mentioned containing new microorganisms.

Example 1

The isolation of human growth hormone mRNA takes place essentially from human pituitary tumor tissue. Five benign pituitary tumors from humans, the Frozen after surgical removal in liquid nitrogen and each weighed 0.4 to 1.5 g, were in 4molar guanidinium thiocyanate solution, the Mercaptoethanol 1molar and buffered to pH 5.0, thawed at 4 ° C and homogenized. The homogenate was over 1.2 ml of 5.7 molar cesium chloride solution containing 100 mmol Ethylenediaminetetraacetic acid, layered and 18 hours at 37,000 revolutions / minute in rotor SW 50,1 one Beckmann ultracentrifuge centrifuged at 15 ° C. It arrived the RNA on the bottom of the centrifuge tube. The Further purification was carried out with a column of oligo-dT and sucrose. Gradient sedimentation. About 10% of the thus isolated RNA coded Growth hormone, as it was estimated, was out of the Incarnation a radioactive amino acid precursor in precipitate from anti-growth hormone in a cell-free translation system from wheat germ; see. Roberts, B.E., and Patterson, B.M., Proc. Nat. Acad. Sci. USA 70, 2330 (1973). This was followed by the production of the cDNA of human growth hormone. The cDNA is fractionated by gel electrophoresis, and that to a about 800 nucleotides in length corresponding position migrating material becomes Cloning selected. This fraction is treated with DNA polymerase I treated, then final Addition of the Hind III linkers. The cDNA is then with the alkaline phosphatase-treated plasmid pBR-322 and DNA ligase recombined. With the recombined DNA E. coli X-1776 is transformed, and a growth hormone DNA  containing strain is selected. This strain is in bred, the DNA is isolated from it, and its nucleotide sequence is determined. The cloned Growth hormone DNA contains the entire amino acid sequence of the human growth hormone encoding nucleotides. The first 23 amino acids of human growth hormone are

The remaining sequence is shown in Table 2:

The process according to the invention makes it the first time possible, one for a specific regulatory protein a higher organism, such as a vertebrate, further isolating the coding nucleotide sequence may be the transfer of the genetic information contained therein be carried out in a microorganism in which unlimited replication is possible. The disclosed method can be applied to the isolation and purification of the human growth hormone gene and its transfer into a microorganism be applied. Disclosed are some new recombined Plasmids which in their nucleotide sequence a Have structure by transcription from a Gene of a higher organism was obtained. Disclosed New microorganisms that are modified become so that they contain a nucleotide sequence which is characterized by Transcription from a gene of a higher organism originated.

Claims (4)

1. DNA transfer vector with a human growth hormone encoding nucleotide sequence obtainable by

• (a) Pituitary cells containing human growth hormone-encoding mRNA isolated,
• (b) extracting the mRNA from the pituitary cells in the presence of an RNase inhibitor composition containing a chaotropic cation, a chaotropic anion, and a disulfide bond-cleaving agent to prevent RNase degradation of the mRNA;
• (c) isolating the mRNA substantially free of protein, DNA and other RNA,
• (d) synthesizing a double-stranded cDNA whose one strand has a mRNA-complementary nucleotide sequence to obtain a cDNA encoding a nucleotide sequence encoding human 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 to the double-stranded cDNA with a nucleotide sequence encoding human growth hormone,

wherein the vector is from plasmid pMB-9 or pBR-332 is derived.   2. DNA transfer vector according to claim 1, characterized in that it transferred to a strain of the microorganism Escherichia coli X-1776 or Escherichia coli RR-1 and there has been replicated. 3. Modified microorganism containing a vector according to Claim 1, characterized in that that it consists of Escherichia coli X-1776 or RR-1.