IE47274B1 - Recombinant dna transfer vector and micro-organism containing a gene from a higher organism - Google Patents

Description

Th·· present invention relates to the iso kit ion ot a specific nucleotide sequence which contains the genetic informal ion coiling for a specific protein, the synthesis of DNA having this specific nucleotide sequence and transfer oi that 1)ΝΛ to a microorganism host wherein Un DNA may be replicated. More specifically, the present invention relates to the isolation <*f the instil in gene and the growth hormone gene, their purification, transfer and replication in a mir-robi.il host and their subsequent characterization. Novel products are produced according to the present invention. These products include a reccsnbinanl plasmid containing file specific nucleotide sequences derived trom a higher oryuniSTft and «ι novel microorganism containing as part of its genetic makeup a specific nuid'Oi ide sequence derived from a higher <»rgunisni.

The symbols and abbreviations used herein are set forth in Ihe following table: DNA - deoxyribonucleic acid RNA - ribonucleic acid eDNA - complementary DNA (enzymatically synthesized from an mRNA sequence) mRNA -- messenger PMA ttlNA - transfer ΚΝΛ dATP - deoxyadenosine triphosphate dGTP - deoxyguanos ine triphosphate. dCTP - deoxycytidine triphosphate The biological significance of the repository of genetic information. It : in DNA is used as a code specifying the A - Adenine T - Thymine G - Guanine C - Cytosine Tris - 2“Aminc-2-hydroxyethvl-1,3-propanediol EDTA - ethylenediamine tetra.icetic acid ATP - adenosine triphosphate TTP - thymidine triphosphate b.ase sequence of DNA, is as a is known that the sequence of bases amino acid sequence of all proteins made by the cell. In addition, portions of the sequence are used for regulatory purposes, to control the timing and amount of each protein made. 7 2 7 4 Tlie nature of these controlling elements is only partially understood. Finally, the sequence of bases in each strand is used as a template for the replication of DNA which accompanies cell division.

The manner by which base sequence information in DNA is used to determine the amino acid sequence of proteins is a fundamental process which, in its broad outlines, is universal to all living organisms. It has been shown that each amino acid commonly found in proteins is determined by one or more trinucleotide or triplet sequences. Therefore, for each protein, there is a corresponding segment of DNA containing a sequence of triplets corresponding to the protein amino acid sequence.

The genetic code is shown in the accompanying table.

In the biological process of converting the nucleotide sequence information into amino acid sequence structure, a first step, termed transcription, is carried out. In this step, a local segment of DNA having a sequence which specifies the protein to be made is first copied with ENA. RNA is a polynucleotide similar to DNA except that ribose is substituted for deoxyribose and uracil is used in place of thymine. The bases in ENA are capable of entering into the same kind of base pairing relationships that exist with DNA. Consequently, the ENA transcript of a DNA nucleotide sequence will be complementary to the sequence copied. Such RNA is termed messenger RNA (mRNA) because of its status as an intermediary between the genetic apparatus and the protein synthesizing apparatus of the cell.

Within the cell, mRNA is used as a template in a complex process involving a multiplicity of enzymes and organelles within the cell, which results in the synthesis of the specific amino acid sequence. This process is referred to as the translation of the mRNA.

There are often additional steps, called processing, which are carried out to convert the amino acid sequence synthesized hy the translational process into a functional protein. An example is provided in the case of insulin.

Genetic Code Phenylalanine(Phe) TTK Iiistidine(His) CAK Leucine(Leu) XTY Glutamine(Gln) CAJ Isoleucine(lle) ATM Asparagine(Asn) ΛΑΚ Methionine(Met) ATC Lysine(Lys) AAJ Valine(Val) GTT. Aspartic acid(Asp) GAK Serine(Ser) qrs Glutamic acid(Glu) GAJ Proline(Pro) CCL Cysteine(Cys) TGK Threonine(Thr) ACL Tryptophan(Try) TGG Alanine(Ala) GCL Arginine(Arg) wcz Tyrosine(Tyr) TAK Glycine(Gly) GGL Termination signal TAJ Termination signal TGA Key: Each 3-letter triplet represents a trinucleotide of i DNA, I 5' end on the left and a 3' end on the right. The letters stan: purine or pyrimidine bases forming the nucleotide sequence.

A = deoxyadenyl G = deoxyguanyl C = deoxycytosyl T = thymidyl X = T or C if Y is A or G X = C if Y is C or T Y = A, G, C or I if X is C Y = A or G if X is T W = C or A if Z is A or G W = C if Z is C or T Z = A, G, C or T if W is C Z = A or G if W is A QR = rc if S is A, G, C or T QR = AG if S is T or C S = A, G, C or T if QR is TC S = T or C if QR is AG J = A or G K = T or C L = A, T, C or G M = A, C or I The immediate precursor of insulin is a single polypeptide, termed proinsulin, which contains the two insulin chains A and B connected by another peptide, C. See Steiner, D.F., Cunningham, D., Spigelman, L. and Aten, B., Science 157, 697 (1967). Recently it has been reported that the initial translation product of Insulin mRNA is not proinsulin itself, but a preproinsulin that contains more than 20 additional amino acids on the amino terminus of proinsulin. See Cahn, S.J., Keim, P. and Steiner, D.F., Proc.Nati.Acad.Scl. USA 73, 1964 (1976) and Lomedico, P.T. and Saunders, G.F., Nucl.Acids Res. 3, 38] (1976). The structure oi the preproinsulin molecule can be represented schematically as H -(pre-peptide)B chain-(C peptide)-A chain-OH. - 4 4727 Many proteins of medical or research significance are found in or made by the cells of higher organisms such as vertebrates. These include, for example, the hormone insulin, other peptide hormones such as growth hormone, proteins involved in the regulation of blood-pressure, and a variety of enzymes having industrial, medical or research significance, it is frequently difficult to obtain such proteins in usable quantities by extraction from the organism, and this problem is especially acute in the case of proteins of human origin. Therefore there is a need for techniques whereby such proteins can be made by cells outside the organism in reasonable quantity. In certain instances, it is possible to obtain appropriate cell lines which can be maintained by the techniques of tissue culture. However, the growth of cells in tissue culture is slow, the medium is expensive, conditions must be accurately controlled, and yields are low. Moreover, it is often difficult to maintain a cultured cell line having the desired differentiated characteristics.

In contrast, microorganisms such as bacteria are relatively easy to grow in chemically defined media. Fermentation technology is highly advanced and can be well controlled. Growth of organisms is rapid and high yields are possible. In addition, certain microorganisms have heen thoroughly characterized genetically and in fact are among the best characterized and best understood organisms.

Therefore it is highly desirable to achieve the transfer of a gene coding for a protein of medical significance, from an organism which normally makes the protein to an appropriate microorgansim. In this way, the protein can be made by the microorganism, under controlled conditions of growth, and obtained in the desired quantities. It is also possible that substantial reductions in the over-all costs of producing the desired protein could be achieved by such a process. In addition, the ability to isolate and transfer the genetic sequence which determines the production of a particular protein into a microorganism having a well-defined genetic background provides a research tool of great value to the study of how the synthesis of such a protein is controlled and how the protein is - 5 ί 7 Ζ 7 ·1 processed alter synthesis. Furthermore, isol itvd genet ic sequences mu·,' be altered to code for variant proteins having altered therapeutic or functional properties.

The present invention provides a means for achieving the above recited goals. A method is disclosed involving a complex series of steps involving enzyme-catalyzed reactions. The nature of these enzyme reactions as they are understood in the prior art is described herewith.

Reverse transcriptase catalyzes the synthesis of DNA complementary to an RNA template strand in the presence of the RNA template, an oligodeoxynucleotide primer and the four deoxynucleoside triphosphates, dATP, dGTP, dCTP, and TTP. The reaction is initiated by the non-covalent bonding of the oligo-deoxynucleotide primer to the 3' end of mRNA followed by stepwise addition of the appropriate deoxynucleotides, as determined by base pairing relationships with the mRNA nucleotide sequence, to the 3' end of the growing chain. The product molecule may be described as a hairpin structure containing the original RNA together with a compleme.ntary strand of DNA joined to it by a single stranded loop of DNA. Reverse transcriptase is also capable of catalyzing a similar reaction using a single stranded DNA template, in which case the resulting product is a double stranded DNA hairpin having a loop of single stranded DNA joining one set of ends. 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 capable of hydrolyzing phosphodiester bonds in double stranded DNA, thereby creating a break in the continuity of the DNA strand. If the DNA is in the form of a closed loop, the loop is converted to a linear structure. The principal feature of an enzyme of this type is that its hydrolytic action is exerted only at a point where a specific nucleotide sequence occurs. Such a sequence is termed the recognition site for the restriction endonuclease. Restriction endonucleases from a variety of sources have been isolated and characterized in terms of the nucleotide sequence of their recognition sites. - 6 Some restriction endonucleases hydrolyze the phosphodiester bonds on both strands at the same point, producing blunt ends. Others catalyze hydroysis of bonds separated by a few nucleotides from each other, producing free single stranded regions at each end of the cleaved molecule.

Such single stranded ends are self-complementary, hence cohesive, and may be used to rejoin the hydrolyzed DNA. Since any DNA susceptible of cleavage by such an enzyme must contain the same recognition site, the same cohesive ends will be produced, so that it is possible to join heterologous sequences of DNA which have been treated with restriction J.0 endonuclease to other sequences similarly treated. See Roberts, R.J., Crit.Rev.Biochem. 4, 123 (1976). Restriction sites are relatively rare, however, the general utility of restriction endonucleases has been greatly amplified by the chemical synthesis of double stranded oligonucleotides bearing the restriction site sequence. Therefore virtually ]_5 any segment of DMA can be coupled to any other segment simply by attaching the appropriate restriction oligonucleotide to the ends of the molecule, and subjecting the prod· ct to the hydrolytic action of the appropriate restriction endonuclease, thereby producing the requisite cohesive ends.

See Heyneker, 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).

SI endonuclease is an enzyme of general specificity capable of hydrolyzing the phosphodiester bonds of single stranded DNA or of single stranded gaps or loops in otherwise double stranded DNA. See Vogt, V.M., Eur.J.Biochem. 33, 192 (1973).

DNA ligase is an enzyme capable of catalyzing the formation of a phosphodiester bond between two segments of DNA having a 5' phosphate and a 3' hydroxyl, respectively, such as might be formed by two DNA fragments held together by means of cohesive ends. The normal function of the enzyme is thought to be in the joining of single strand nicks in an otherwise double stranded DNA molecule. However, under appropriate conditions, DNA -7-. 2 7-1 ligase is capable of catalyzing blunt end ligation in which two molecules having blunt ends are covalently joined. See Sgaramella, V., Van do Sande, J.H., and Khor.ina, H.G., Proc.N.it i .Acad.Sci.__USA 67, 1468 (1970) .

Alkaline phosphatase is an enzyme of general specificity capable of hydrolyzing phosphate esters Including 5' terminal phosphates on DNA.

A further step in the overall process to be described is the insertion of a specific DNA fragment into a DNA vector, such as a plasmid. Plasmid is the term applied to any autonomously replicating DNA unit which might be found in a microbial cell, other than the genome of the host cell itself. A plasmid is not genetically linked to the chromosome of the host cell. Plasmid DNA's exist as double stranded ring molecules generally on the order of a few million molecular weight, although some are greater than g molecular weight, and they usually represent only a small percent of the total DNA of the cell. Plasmid DNA is usually separable from host cell DNA by virtue of the great difference in size between them.

Plasmids can replicate independently of the rate of host cell division and in some cases their replication rate can be controlled by the investigator by variations in the growth conditions. Although the plasmid exists as a closed ring, it is possible by artificial means to introduce a segment of DNA into the plasmid, forming a recombinant plasmid with enlarged molecular size, without substantially affecting its ability to replicate or to express whatever genes it may carry. The plasmid therefore serves as a useful vector for transferring a segment of DNA into a new host cell.

Plasmids which are useful for recombinant DNA technology typically contain genes which may be useful for selection purposes, such as genes for drug resistance.

To illustrate the practice of the present invention, the isolation and transfer of the rat insulin gene is described in detail, insulin was chosen for this effort because of its central significance from the standpoint of clinical medicine, and from the standpoint of basic research. The disclosed procedure is applicable by those of ordinary skill in the art to the isolation of the insulin gene of other organisms, including humans. - 8 Insulin was first isolated in 1922. At the present time, the use of this hormone in the treatment of diabetes is well-known. Although slaughterhouses provide beef and pig pancreases as insulin sources, a shortage of the hormone is developing as the number of diabetics increases worldwide. Moreover, some diabetics develop an allergic reaction to beef and pig insulin, with deleterious effects. Ihe ability to produce human insulin in quantities sufficient to satisfy world needs is therefore highly desirable. Manufacturing human insulin in bacteria is a technique which could achieve this desired goal. However, prior to the present invention, progress toward this desired goal has been thwarted by the fact that no technique has been developed to introduce the insulin gene into a bacteria. The present invention provides such a technique.

The ability to obtain DNA having a specific sequence which is the genetic code for a specific protein makes it possible to modify the nucleotide sequence by chemical or biological means such that the specific protein ultimately produced is also modified. This would make it possible to produce, for example, a modified insulin tailored to suit a specific medical need. The genetic capacity to produce any insulin-related amino acid sequence having the essential functional properties of insulin may therefore be conferred upon a microorganism. Similar considerations apply in the case of growth hormones.

The ability to transfer the genetic code for a specific protein necessary to the normal metabolism of a particular higher organism to a microorganism such as a bacterium opens significant possibilities for culture production of such proteins. This in turn affords significant possibilities for augmenting or replacing the output of such proteins with those produced by microorganisms altered pursuant to this invention, whenever the ability of the higher organism to function normally in the production of such proteins has been impaired, and suggest, £, the possibility of establishing symbiotic relationships between microorganisms produced pursuant to this invention and human heings with chronic or acute deficiency diseases, whereby microorganisms genetically altered as herein - 9 4 ? Ζ 7 J taught might be implanted in or otherwise associated with a human to compensate for the pathologic deficiency in the metabolism of the latter.

Thus, according to the present invention there is provided a DNA transfer vector containing within its nucleotide sequence a subsequence having the structure of, and transcribed from, a gene of a higher organism.

More specifically, the invention provides a microorganism modified to contain a nucleotide sequence having the structure of, and transcribed from, a gene of a higher organism.

The invention also provides a method of making a DNA transfer vector having a nucleotide sequence coding for a gene of a higher organism comprising: (a) isolating cells containing mRNA coding for the desired protein, (b) extracting the mRNA from the cells, if necessary in the presence of an RNase inhibitor to prevent RNase degradation of the mRNA, (c) separating the mRNA substantially free of protein, DNA and other RNA, (d) synthesising a double stranded cDNA wherein one strand has a nucleotide sequence complementary to that of the mRNA, thereby producing cDNA having a nucleotide sequence coding for the desired protein, (e) providing a DNA transfer vector having reactant ends capable of being joined together or of being joined with double strands DNA, and (f) joining the DNA transfer vector with the double stranded cDNA having a nucleotide sequence coding for the desired protein, whereby a DNA transfer vector having a nucleotide sequence coding for the desired protein is produced.

In the DNA transfer vector of the invention the gene of a higher organism may code for insulin. Thus, for example, the DNA transfer vector may comprise a nucleotide sequence coding for the A chain of human insulin comprising:51---GGL^ ATM2GTL3GAJ4CAJ5TGKeTGK7ACL8QR9SgATM10TGK11QR12S1?X13TY13 TAK14GAJ15X16TY16GAJ17AAK18TAK19TGK2OAAK21---3‘.

Such a DNA transfer vector may comprise an additional nucleotide sequence coding for the B chain of human insulin comprising: 5 '---TTK^GTLjAAKjGAJ^AKgXgTYgTGI^GGLgQRgSgCAK.^ Χ11ΤΥΐ16ΤΒ1§Ασΐ3Ε“ΐ4Χ15ΤΥ15ΤΑΚ16Χ17ΤΪ17ΰΤ£18Τ<3Κ196512Ο6Α321 W22GZ22GGL23TTK24TTK25TAK26aCI'27CCL28AAJ29ACL3O—3' Also, the nucleotide sequence coding for the A and B chains of human insulin may be joined in the partial sequence '---ACL3oGCL---3' and from 1 to 100 nucleotide triplets of sequence (Na Nb Nc), wherein Na, Nb and Nc may be A, T, G or C, may be interposed in continuous sequence between said ACL3Q and GCL^ provided that Na Nb Nc is neither TAJ nor TGA.

The DNA transfer vector of the invention may be transferred to and replicated in a micro-organism strain to provide a micro-organism modified according to the invention.

In one preferred aspect the invention provides a method of making a DNA transfer vector having a nucleotide sequence coding for insulin comprising: (a) isolating islet cells containing mRNA coding for insulin from the pancreas of an insulin-producing organism, (b) extracting the mRNA from the islet cells in the presence of an RNase inhibitor composition, whereby RNase degradation of the mRNA is prevented. 4 7 2 7 I (c) separating the mRNA substantially free of protein, DNA and other RNA, (d) synthesizing a double stranded cDNA wherein one strand has a nucleotide sequence complementary to that of the mRNA, thereby producing cDNA having a nucleotide sequence coding for insulin, (e) providing a DNA transfer vector having reactant ends capable of being joined together or of being joined with double stranded cDHA, and (f) joining the DNA transfer vector with the double stranded cDNA having a nucleotide sequence coding for insulin, whereby a DNA transfer vector having a nucleotide sequence coding for insulin is produced.

Specifically such a method may be one for making a micro-organism having a nucleotide sequence coding for insulin in which subsequent to step (f) the thus-produced DNA transfer vector joined with cDNA having a nucleotide sequence coding for insulin is mixed with a micro-organism.

In another preferred aspect the invention provides a method of making a DNA transfer vector having a nucleotide sequence coding for growth hormone comprising: (a) isolating pituitary cells containing mRNA coding for growth hormone; (b) extracting the mRNA from the pituitary cells in the presence of an RNase inhibitor composition, whereby RNase degradation of the mRNA is prevented, (c) separating the mRNA substantially free of protein, DNA and other RNA, (d) synthesizing a double stranded cDNA wherein one strand has a nucleotide sequence complementary to that of the mRNA, thereby producing cDNA having a nucleotide sequence coding for growth hormone, (β) providing a DNA transfer vector having reactant ends capable of being joined together or of being joined with double stranded cDNA, and (f) joining the DNA transfer vector with the double 5 stranded cDNA having a nucleotide sequence coding for growth hormone, whereby a DNA transfer vector having a nucleotide sequence coding for growth hormone is produced.

Again, specifically such a method may be one for making a micro-organism having a nucleotide sequence coding for growth hormone in which subsequent to step (f) the thus-produced DNA transfer vector joined with cDNA having a nucleotide sequence coding for growth hormone is mixed with a micro-organism. '727 1 Thus, there are provided methods for isolating a specific nucleotide sequence containing gt^netic information, synthesis of DNA having the specific nucleotide sequence and transfer of the DNA to a host microorganism.

The invention is exemplified by specific DNA sequences transferred to a bacterium including the structural gene for rat preproinsulin, the gene for rat growth hormone and the gene for human growth hormone. It is therefore contemplated that the method is of good applicability to the transfer of any desired DNA sequence from a higher organism, such as a vertebrate, to any microorganism. A higher organism is here defined as any eucaryotic organism having differentiated tissues, including but not limited to, insects, molluscs, plants, vertebrates including mammals, the latter category including cattle, swine, primates and humans. A microorganism, as is understood in the art, may be any microscopic living organism such as is included in the term, protist, whether procaryotic or eucaryotic, including for example bacteria, protozoa, algae and fungi, the latter category including yeasts. More specifically according to the invention, a selected cell population is first isolated by an improved irethod. Intact mRNA is extracted from the cells by a novel procedure whereby virtually all RNAse activity is suppressed. Intact messenger RNA is purified from the extract by column chromatography and subjected to the action of the enzyme reverse transcriptase acting in the presence of the four deoxynucleoside triphosphates needed to synthesize a complementary (cDNA) strand. The product of this first reaction with reverse transcriptase is subjected to a procedure which selectively removes the ribonucleotide sequence. The remaining deoxynucleotide sequence, complementary to the original mRNA, is incubated in a second reaction with reverse transcriptase or DNA polymerase in the presence of the four deoxynucleoside triphosphates. The resulting product Is a duplex cDNA structure having its complementary strands joined together at one end by a single stranded loop. This product is then treated with single strand specific nuclease which cleaves the single stranded loop. The resulting double stranded cDNA is next extended in length by the addition at both ends of a specific DNA containing a restriction enzyme recognition site sequence. The addition is catalyzed by a DNA ligase enzyme. The extended cDNA is next treated with a restriction endonuclease, producing self-complementary single stranded ends at the five-prime termini of each strand in the duplex.

A plasmid DNA having a recognition site for the same restriction endonuclease is treated with the enzyme, in order to cleave the polynucleotide strand and produce self-complementary single strand nucleotide sequences at the 5' termini. The 5' terminal phosphate groups on the single stranded ends are removed to prevent the plasmid from forming a ring structure capable of transforming a host cell. The prepared cDNA and plasmid DNA are incubated together in the presence of DNA ligase. Under the reaction conditions described, the formation of a viable closed ring of plasmid DNA can only occur if a segment of cDNA is included. The plasmid containing the cDNA sequence is then introduced into an appropriate host cell. Cells which have received a viable plasmid are detected by the appearance of colonies having a genetic trait conferred by the plasmid, such as drug resistance. Pure bacterial strains containing the recombinant plasmid having the incorporated cDNA sequence are then grown up, and the recombinant plasmid reisolated. Large amounts of recombinant plasmid DNA may he prepared in this manner and the specific cDNA sequence reisolated therefrom by endonucleolytic cleavage with the appropriate restriction enzyme.

The method of the invention is applicable to the isolation and transfer to a host microorganism of any desired nucleotide sequence obtained from a higher organism, including man. The method will be useful in the transfer of a gene coding for a specific protein having medical or industrial value and in the microbiological synthesis of such protein. In demonstrating the invention, the nucleotide sequence coding for insulin has been isolated from rat, tranferred to bacteria and replicated therein. Similarly, the nucleotide sequence coding for rat growth hormone has been isolated, transferred and replicated in bacteria. The method is applicable to the transfer of a nucleotide sequence isolated from a human source, including human - 15 4 'i 2 7 1 insulin and growth hormone, as well as other polypeptide hormones.

The sequence of steps comprising the method of the invention can be classified in four general categories: 1. The isolation of a desired cell population from a higher organism.

There are two potential sources of a genetic sequence coding for specific protein: the DNA of the source organism itself, and an RNA transcript of the DNA. The current safety requirements in the United States of the National Institutes of Health specify that human genes of any kind can be put into recombinant DNA, and then into bacteria, only after the genes have been very carefully purified or in special high-risk (P4) facilities. See Federal Register, Vol. 41, No. 131, July 7, 1967, pp. 27902-27943. Therefore, for any procedure having potential utility for the production of the human protein, such as the present method the preferred approach is the isolation of specific mRNA having a nucleotide sequence which codes for the desired protein. The adoption of this strategy has the further advantage that the mRNA can be more easily purified than DNA extracted from the cell.

In particular, it is possible to take advantage of the fact that in highly differentiated organisms such as vertebrates, it may be possible to identify a specific population of cells having a specific location within the organism, whose function is primarily devoted to the production of the protein In question. Alternatively, such a population may exist during a transient developmental stage of the organism. In such cell populations, a large portion of the mRNA Isolated from the cells will have the desired nucleotide sequence. Therefore, the choice of cell population to be isolated, and the method of isolation employed, can be substantially advantageous from the standpoint of the initial purity of the mRNA isolated therefrom.

In most tissues, glands and organs, the cells are held together by a generally fibrous network of connective tissue, composed principally of - 16 collagen but which may also include, depending on the tissue, other structural proteins, polysaccharides and mineral deposits. The isolation of cells from a given tissue necessarily requires the employment of techniques for releasing the cells from the connective tissue matrix. The isolation and purification of a specific differentiated cell type therefore embodies two major stages: the separation of cells from the connective tissue matrix, and the separation of cells of the desired type from all other cells types found in the tissue. The operating principles inherent and disclosed in the present invention will be applicable to the isolation of a variety of cell types from a variety of tissue sources. By way of example, the isolation of islets of Langerhans from pancreas, suitable for the isolation of mRNA coding for insulin, will be described.

Insulin-producing cells may be derived from other sources, such as foetal calf pancreas or cultured islet tumor cells. The isolation of pure islet cells will be much simpler in such cases, especially where pure cell cultures are used. The method of isolating islet cells discussed supra would not be needed in such cases, however, the method remains advantageous because of its general applicability.

Frequently, it will he found that the proportion of desired mRNA can be increased by taking advantage of cellular responses to environmental stimuli. For example, treatment with a hormone may cause increased production of the desired mRNA. Other techniques include growtii at a particular temperature and exposure to a specific nutrient or other chemical substance. In the isolation of rat growth hormone mRNA, treat25 ment of cultured rat pituitary cells with thyroid hormone and glucocorticoids synergistically raised the proportion of growth hormone mRNA by a significant amount. 2. Extraction of mRNA. An important feature of the present invention is the essentially complete removal of RNase activity in the cell extract.

The mRNA to be extracted is a single polynucleotide strand, unpaired with any complementary strand. Therefore, the hydrolytic cleavage of a single phosphodiester bond in the sequence would render the entire molecule useless - 17 4 7 2 7 4 for the purpose of transferring an intact genetic sequence to a microorganism. As stated hereinabove, the enzyme RNase is widely distributed, active and exceptionally stable. It is found on the skin, survives ordinary glassware washing techniques and sometimes contaminates stocks of organic chemicals. The difficulties are especially acute in dealing with extracts of pancreas cells, since the pancreas is a source of digestive enzymes and is, therefore, rich in RNase. However, the problem of RNase contamination is present In all tissues, and the method disclosed herein to eliminate RNase activity is applicable to all tissues. The exceptional effectiveness of the method is demonstrated in the present invention by the successful isolation of intact mRNA from isolated islet cells of the pancreas.

The present invention preferably employs in combination a chaotropic anion, a chaotropic cation and a disulfide bond-breaking agent, during cell disruption and during all operations required to separate RNA essentially free from protein. The effectiveness of the combined action of the foregoing agents has been demonstrated by their use in the isolation of essentially undegraded mRNA in good yield from isolated islets of Langerhans of rat pancreas.

Choice of suitable chaotropic ions is based upon their solubility in aqueous media and upon availability. Suitable chaotropic cations include guanidinium, carbamoylguanidinium, guanylguanidinium, and lithium.

Suitable chaotropic anions include iodide, perchlorate, thiocyanate, diiodosalicylate. The relative effectiveness of salts formed by combining such cations and anions will be determined in part by their solubility. For exanple, lithium diiodosalicylate is a more potent denaturant than guanidinium thiocyanate, but it has a solubility of only about 0.1M and is also relatively expensive. Guanidinium thiocyanate provides the preferred cation-anion combination, because it is readily available and is highly soluble in aqueous media, up to 5M. Thus, for example, the RNase inhibitor composition may comprise 4M guanidinium thiocyanate.

Thiol conpounds, such as ?-mercaptoethanol, are known to break intramolecular disulfide bonds in proteins by a thiol-disulfide interchange - 18 47Ζί4 reaction. Many thiol canwunds are knram to be effective, including besides ij-mercaptoethanol, dithiothreitol, cysteine, propanol, and Himorraptan. Aqueous solubility in a necessary property, since the thiol compound must be present in large excess over the intra5 molecular disulfides, in order to drive the interchange reaction essentially to completion, β-mercaptoethanol is preferred because of its ready availability at reasonable price.

For the purpose of inhibiting RNase during extraction of RNA from cells or tissues, the effectiveness of a given chaotropic salt is directly related to its concentration. The preferred concentration is therefore the highest concentration that can be employed, as a practical matter.

The success of the present invention in preserving mRNA intact during extraction is thought to depend upon the rapidity with which the RNase is denatured, in addition to the extent of denaturation. This is thought to explain, for example, the superiority of guanidinium thiocyanate over the hydrochloride, despite the fact that the hydrochloride is only slightly less potent as a denaturing agent. The effectiveness of a denaturant is defined as the threshold concentration needed to achieve complete denaturation of a protein. On the other hand, the rate of denaturation of many proteins is dependent on the denaturant concentration, relative to the threshold, raised to from the 5th to the 10th power. See Tanford, C.A. Adv.Prot.Chem. 23, 121 (1968). Qualitatively this relationship suggests that a denaturant only slightly more potent than guanidinium hydrochloride can denature a protein many times more rapidly at the same concentration.

The relationship between the kinetics of RNase denaturation and the preservation of mRNA during its extraction from cells is not thought to have been recognized or exploited, prior to the present invention. The foregoing analysis, if correct, suggests that the preferred denaturant will be one having a low threshold denaturing concentration combined with a high aqueous solubility. For this reason, guanidinium thiocyanate is preferred over lithium diiodosalicylate even though the latter is a more potent denaturant, because of solubility of guanidinium thiocyanate - 19 4 7 2? 4 is much greater, hence it can bo used at a concentration permitting more rapid RNase inactivation. The foregoing analysis also explains why guanidinium thiocyanate is preferred over the comparably soluble hydrochloride salt, since the former is a somewhat more potent denaturant.

The use of a disulfide bond breaking agent in combination with a denaturant potentiates and enhances the effectiveness of the latter by permitting the RNase molecule to become completely unfolded. The thiol compound is thought to enhance the forward rate of the denaturation process by preventing the rapid renaturation which can occur when the intramolecular disulfide bonds are left intact. Furthermore, any contaminating RNase remaining in the mRNA preparation will remain substantially inactive, even in the absence of the denaturant and thiol. Disulfide bond breaking agents having thiol groups will be effective to some extent at any concentration, although generally speaking, a large excess of thiol groups to intramolecular disulfide bonds is preferred to drive the interchange reaction in the direction of intramolecular disulfide cleavage. On the other hand, many thiol compounds are malodorous and unpleasant to work with in high concentration, so that a practical upper concentration limit ,> exists. Using 5“mercaptoethanol, concentrations in the range from 0.05M to 1.0M have been found effective, and 0.2M is considered optimal, for the isolation of undegraded in RNA from rat pancreas.

The pH of the medium during extraction of mRNA from cells may be in the range of pH 5.0 - 8.0.

Following the cell disruption step, the RNA is separated from the bulk of the cellular protein and DNA. A variety of procedures has been developed for this purpose, any of which is suitable, all of which are well-known in the art. A common practice in the prior art is to use an ethanol precipitation procedure which selectively precipitates RNA. The preferred technique of the present invention is to bypass the precipitation step and layer the homogenate directly on a solution of 5.7 11 cesium chloride in a centrifuge tube and then to subject the tube to centrifugation as described in Glisin, V., Crkvenjakov, R., and Byus, C., Biochemistry 13, 2633 (1974). This method is preferred because an environment continuously hostile to RNase is maintained and RNA Is recovered in high yield, free of DNA and protein. - 20 The above recited procedures result in the purification of total RNA from the cell homogenate. However, only a portion of such RNA is the desired mRNA. Xn order to further purify the desired mRNA, advantage is taken of the fact that in the cells of higher organisms, mRNA, after transcription, is further processed in the cell by the attachment of polyadenylic acid. Such mRNA containing poly A sequences attached thereto may he selectively isolated by chromatography on columns of cellulose to which is attached oligo-thymidylate, as described hy Aviv, H., and Leder, P., supra. The foregoing procedures are sufficient to provide essentially pure, intact, translatable mRNA from sources rich in RNase.

The purification of mRNA and subsequent in vitro procedures may be carried out in essentially the same manner for any mRNA, regardless of the source organism.

Under certain circumstances, for example when tissue culture cells are used as the mRNA source, RNase contamination may be sufficiently low that the RNase inhibition method just described will not be needed.

In such cases, prior art techniques for reducing RNase activity may be sufficient. 3. Formation of cDNA. Reference is made to the accompanying drawings for a schematic representation of the remaining steps of the method. The first in these remaining steps is the formation of a sequence of DNA cornplemsntary to the purified mRNA. The enzyme of choice for this reaction is reversed. transcriptase, although in principle any enzyme capable of forming a faithful complementary DNA strand using the mRNA as a template could be used.

The reaction may be- carried out under conditions described in the prior art, using mRNA as a template and a mixture of four deoxynucleoside triphosphates as precursors for the DNA strand. Xt is convenient to provide that one 32 of the deoxynucleoside triphosphates be labelled with P in the alpha position in order to monitor the course of the reaction, provide a tag for recovering the product after separation procedures such as chromatography and electrophoresis, and for the purpose of making quantitative estimates of recovery. See Efstratiadis, A., et al., supra. - 21 4 7 2 7 -1 As shewn in the drawing, the product of the reverse transcriptase reaction is a double stranded hairpin structure with a non-covalent linkage between the RNA strand and the DNA strand.

The product of the reverse transcriptase reaction is removed from the reaction mixture by standard techniques known in the art. It has been found useful to employ a combination of phenol extraction, chromatography 1/ on Sephadex G-100 and ethanol precipitation.

Once the cDNA has been enzymatically synthesized, the RNA template may be removed. Various procedures are known in the prior art for the selective degradation of RNA in the presence of DNA. The preferred method is alkaline hydrolysis, which is highly selective and can be readily controlled by pH adjustment.

Following the alkaline hydrolysis reaction and subsequent neutralization, 32 the P labeled cDNA may be concentrated by ethanol precipitation if desired.

Synthesis of a double stranded hairpin cDNA is accomplished by the use of an appropriate enzyme, such as DNA polymerase or reverse transcriptase. Reaction conditions similar to those described previously are employed, including the use of an <£ -32p labeled nucleoside triphosphate. Reverse transcriptase Is available fran a variety of sources. A convenient source is avian myeloblastosis virus. The virus is available from Dr. D. J.

Beard, Life Sciences Incorporated, St. Petersburg, Florida, who produces the virus under contract with the National Institute of Health.

Following the formation of the cDNA hairpin, it may be convenient to purify the DNA from Che reaction mixture. As described previously, it lias been found convenient to employ the steps of phenol extraction, chromatography on Sephadex G-100 and ethanol precipitation to purify the DNA product free of contaminating protein.

The hairpin structure may be converted to a conventional double stranded DNA structure by the removal of the single stranded loop joining the ends of the complementary strands. A variety of enzymes capable of specific hydrolytic cleavage of single stranded regions of DNA is available for this purpose. A convenient enzyme for this purpose is the 1/ Trade Mark, Pharmacia Inc., Uppsala, Sweden. - 22 727 4 SI nuclease isolated from Aspergillus oryzae. The enzyme may be purchased from Miles Research Products, Elkhart, Indiana. Treatment of the hairpin DNA structure with SI nuclease results in a high yield of cDNA molecules with base paired ends. After the extraction, chromatography and ethanol precipitation as previously described. The use of reverse transcriptase and SI nuclease in the synthesis of double stranded cDNA transcripts of mRNA has been described by Efstratiadis, et al., supra.

Optionally, the proportion of cDNA molecules having blunt ends may be maximized by treatment with Escherichia coli DMA. polymerase I in the presence of the four deoxynucleoside triphosphates, The combination of the enzyme’s exonuclease and polymerase activities acts to remove any 3' protruding ends and to fill any 5' protruding ends. Participation of the maximum proportion of cDNA molecules in the subsequent ligation reactions is thereby assured.

The next step in the process involves the treatment of the ends of the cDNA product to provide appropriate sequences at each end containing a restriction endonuclease recognition site. The choice of DNA fragment to be added to the ends is determined by matters of manipulative convenience.

The sequence which is to be added to the ends is chosen on the basis of the 2o particular restriction endonuclease enzyme chosen, and this choice in turn depends on the choice of DNA vector with which the cDNA is to be recombined. The plasmid chosen should have at least one site susceptible to restriction endonuclease cleavage. For example, the plasmid pMB9 contains one restriction site for the enzyme Hind III. Hind III is isolated from Hemophilus influenzae and purified by the method of Smith, H.O., and Wilcox, K.W., J.Mol.Biol. 51, 379 (1970). Tho enzyme, Hae III, Γrom Hemuplii I us aegyptious is purified by the method of Middleton, J.H., Edgell, M.H., and Hutchison III, C.A., J.Virol. 10, 42 (1972). An enzyme from Hemophilus suis, designated Hsu I, catalyzes the same site-specific hydrolysis, at 3q the same recognition site, as Hind III. These two enzymes are therefore considered as functionally interchangeable. - 23 4 7 2 7 4 It is convenient to employ a chemically synthesized double stranded decanucleotide containing the recognition sequence for Hind III, for the purpose of attachment to the ends of the cDNA duplex. The double stranded decanucleotide has the sequence shown in Figure X. See Heyneker, H.L., et al., and Scheller, R.H., et al., supra. A variety of such synthetic restriction site sequences is available to workers in the art, so that it is possible to prepare the ends of a duplex DNA so as to be sensitive to the action of any of a wide variety of restriction endonucleases.

The attachment of restriction site sequences to the ends of cDNA may be accomplished by any method known to workers in the art. The method of choice is a reaction termed blunt end ligation, catalyzed by DNA ligase purified by the method of Panet, A., et al., Biochemistry 12, 5045 (1973).

The blunt end ligation reaction has been described hy Sgaramella, V., et al., supra. The product of the blunt end ligation reaction between a blunt ended cDNA and a large molar excess of double stranded decanucleotide containing the Hind III endonuclease restriction site is a cDNA having Hind III restriction site sequences at each end. Treatment of the reaction product with Hind III endonuclease results in cleavage at the restriction site with the formation of single stranded 5' self-complementary ends, as shown in figure 1. 4. Formation of a recombinant DNA transfer vector. In principle, a wide variety of viral and plasmid DNA's could be used to form recombinants with a cDNA prepared in the manner just described. The principal requirements are that the DNA transfer vector be capable of entering a host cell, undergoing replication in the host cell and should, in addition, have a genetic determinant through which it is possible to select those host cells which have received the vector. For reasons of public safety, however, the range of choice should be restricted to those transfer vector species deemed suitable for the type of experiments employed, according with the NIH guidelines, supra. The list of approved DNA transfer vectors is continuously being enlarged, as new vectors are developed and approved by the NIH Recombinant DNA Safety Committee, and it is to be understood that - 24 47274 this invention contemplates the use of any viral and plasmid DNA's that have the described capabilities, including those on which NIH approval may later be granted. Suitable transfer vectors which are currently approved for use include a variety of derivatives of bacterio5 phage lambda (See e.g. Blattner, F.R., Williams, B.G., Blechl, A.E., Denniston-Thompson, K., Faber, H.E., Furlong, L.A., Grunwald, D.J., Kiefer, D.O., Moore, D.D., Schumm, J.W., Sheldon, E.L., and Smithies, 0., Science 196, 161 (1977) and derivatives of the plasmid col El, see e.g. Rodriguez, R.L., Bolivar, S., Goodman, H.M., Boyer, H.W., and Betlach, M.N.: IO ICN-UCLA Symposium on Molecular Mechanisms In The Control of Gene Expression, D.P. Nierlich, W.J. Rutter, C.F. Fox, Eds. (Academic Press, HY, 1976), pp. 471-477. Plasmids derived from col El are characterized by being relatively small, having molecular weights of the order of a few million, and having the property that the number of copies of plasmid DNA per host cell can be increased from 20-40 under normal conditions to 1000 or more, by treatment of the host cells with chloramphenicol. The ability to amplify the gene dosage within the host cell makes it possible under appropriate circumstances, under the control of the investigator, to cause the host cell to produce primarily proteins coded for hy genes carried on the plasmid. Such derivatives of col El are therefore preferred transfer vectors in the process of the present invention. Suitable derivatives of col El include the plasmids pMB-9, carrying the gene for tetracycline resistance, and pBR-313, pBR-315, pBR-316, pBR-317 and pBR-322, which contain, in addition to the tetracycline resistance gene, a gene for amplicillin resistance. The presence of the drug resistance genes provides a convenient way for selecting cells which have been successfully infected by the plasmid, since colonies of such cells will grow in the presence of the drug whereas cells which have not received the plasmid will not grow or form colonies. In the experiments described herein as specific examples of the present invention, a plasmid derived from col El was used throughout, containing, in addition to the described drug resistance marker, one Hind III site.

As with the choice of plasmid, the choice of a suitable host is in principle very broad hut for purposes of public safety, narrowly restricted. - 25 *« 7 2 7 -1 A strain of E. coli designated X-1776 has been developed and has received NIH approval for processes of the type described herein, using P2 containment facilities. See Curtiss, III, R., Ann.Rev.Hicrobiol·., 30, 507 (1976). E. coli RR-1 is suitable where P3 containment facilities are available. As in the case of the plasmids, it will be understood that the invention contemplates use of any host cell strains having the capability of acting as a transferee for the chosen vector, including protists other than bacteria, for example yeasts, whenever such strains are approved for use under the NIH guidelines.

Recombinant plasmids are formed by mixing restriction endonucleasetreated plasmid DNA with cDNA containing end groups similarly treated. In order to minimize the chance that segments of cDNA will form combinations with each other, the plasmid DNA is added in molar excess over the cDNA.

In prior art procedures this has resulted in the majority of plasmids circularizing without an inserted cDNA fragment. The subsequently transformed cells contained mainly plasmid and not cDNA recombinant plasmids. As a result, the selection process was very tedious and time consuming. The prior art solution to this problem has been to attempt to devise DNA vectors having a restriction endonuclease site in the middle of a suitable marker gene such that the insertion of a recombinant divides the gene thereby causing loss of the function coded by the gene.

Preferably, a method for reducing the number of colonies to be screened for recombinant plasmids is employed. The method involves treating plasmid DNA cut with the restriction endonuclease with alkaline phosphatase, an enzyme commercially available from several sources, such as Worthington Biochemical Corporation, Freehold, New Jersey. Alkaline phosphatase treatment removes the 5’-terminal phosphates from the endonuclease generated ends of the plasmid and prevents self-ligation of the plasmid DNA. Consequently, circle formation, hence transformation, will be dependent on the insertion of a DNA fragment containing 5'phosphorylated termini. The described process reduces the relative frequency of transformation in the absence of recombination to less than 1 to 10+Z|.

The present Invention is based on the fact that the DNA ligase catalyzed reaction takes place between a 5'-phosphate DNA end group - 26 4 7 274 and a 3'-hydroxyl DNA end group. If the terminal 5'-phosphate is removed, no joining reaction occurs. Where double-stranded DNA is to be joined, three situations are possible, as shown in Table 1.

TABLE 1 Xn Table 1, double-stranded DNA is schematically represented by solid parallel lines, while their respective 5' and 3' end groups are labeled with hydroxyi (OH) or phosphate (OPO-jHj), as the case may be. In case I, ' phosphates occur on both reactant ends with result that both strands become covalently joined. In case II only one of the strands to he joined has a terminal 5' phosphate, with the result that a covalent single-strand linkage occurs leaving a single-strand break of discontinuity on the other strand. The strand not covalently joined remain associated with the joined molecule by virtue of the hydrogen bonding interactions between complementary base-pairs on opposite strands, as is well known in the art. In case III neither of the reactant ends has a 5'-phosphate and no joining reaction can occur.

Unwanted joining reactions can he prevented therefore by treatment of the appropriate reactant ends, whose joining is to be prevented, to remove the 5'-phosphate groups therefrom. Any method suitable for removal of 5'-phosphate groups that does not otherwise damage the DNA structure may be employed. Hydrolysis catalyzed by the enzyme alkaline phosphatase is preferred. - 27 YV.17 4 The process just described is also useful in the situation where a linear DNA molecule is to be cleaved into two .sub-fragments, typically with the use of a restriction endonuclease enzyme, then reconstituted in the original sequence. The sub-fragments may be separately purified and the desired sequence may be reconstituted by rejoining the sub-fragments. The enzyme DNA ligase, which catalyzes the end-to-end joining of DNA fragments, may be employed for this purpose. See Sgaramella, V., Van de Sande, J.H., and Khorana, H.G., Proc.Natl.Acad.Sci USA 67, 1468 (1970).

Where the sequences to be joined are not blunt-ended, the ligase obtained from Ii. coli may be used, Modrich, P., and Lehman, I.R,, J.Biol.Chem. 245. 3626 (1970).

The efficiency of reconstituting the original sequence from subfragments produced by restriction endonuclease treatment will be greatly enhanced by the use of a method for preventing reconstitution in improper sequence. This unwanted result is prevented by treatment of the homogeneous length cDNA fragment of desired sequence with an agent capable of removing the 5'-terminal phosphate groups on the cDNA prior to cleavage of the homogeneous cDNA with a restriction endonuclease. The enzyme, alkaline phosphatase, is preferred. The 5'-terminal phosphate groups are a structural prerequisite for the subsequent joining action of DNA ligase used to reconstitute the cleaved sub-fragments. Therefore, ends which lack a 5'-terminal phosphate cannot be covalently joined. The DNA sub-fragments can only be joined at the ends containing a 5'-phosphate generated by the restriction endonuclease cleavage performed on the isolated DNA fragments.

The foregoing method prevents the formation of the most significant unwanted joining reaction, namely the joining of the two fragments in reverse sequence, back-to-front instead of front-to-back. Other possible side reactions, such as dimer formation and cyclization are not prevented, since these can occur by a reaction of type XI, supra, Table 1. Such side reactions are less troublesome, however since they lead to physically separable and identifiable products, whereas recombination in reverse order does not. - 28 For the purpose of illustrating the above described procedures, cDNA coding for rat insulin has been isolated and recombined with a plasmid. The DNA molecules were used to transform E. coli X-1776.

Transformants were selected by growth on medium containing tetracycline.

One recombinant plasmid DNA obtained from transformed cells was found to contain an inserted DNA fragment approximately 410 nucleotides in length. Other recombinants, isolated by similar procedures, were also obtained and analyzed. The inserted fragments were released from the plasmid hy Hind III or HSU I endonuclease digestion and were subjected to DNA sequence analysis by the method of Maxam, A.M. and Gilbert, W., Proc.

Natl.Acad.Sci USA 74, 560 (1977). The nucleotide sequences of the inserted DNA fragments were found to be overlapping and to contain the entire coding region for rat proinsulin I, as well as 13 out of 23 amino acids of the prepeptide sequence. A composite of the nucleotide sequence of this region was constructed as shown hereinafter.

In a similar procedure, cDNA coding for rat growth hormone was isolated recombined with a plasmid and transferred to I!, coli. A nucleotide sequence approximately 800 nucleotides in length was reisolated after extensive replication in E. coil and found to comprise the entire coding sequence for RGH as well as portions of the precursor peptide and a portion of the 5' untranslated region.

The method just described is generally applicable to the isolation and purification of a gene from a higher organism, including a human gene, and its transfer to and replication in a microorganism. Novel recombinant plasmids containing all or a portion of the isolated gene are described.

Novel microorganisms hitherto unknown in nature are described, having as part of their genetic makeup a gene from a higher organism. Specific examples detailing each step of the process as applied to the isolation, purification and transfer of the rat insulin gene into E. coli will next be described, in order to more clearly reveal the characteristics and utility of the invention. The following examples characterize recombinant plasmids containing portions of the rat insulin gene, the rat growth hormone gene, the human insulin gene and the human growth hormone gene and microorganisms containing said genes. - 29 i 7 2 Ί Example 1 The described procedures demonstrate the extraction and isolation of rat unsulin mRNA, the synthesis of a DNA complementary thereto and the characterization of the complementary DNA. To prepare purified rat islet cells, the pancreas of an anesthetized rat was infused with Hank's salt solution by retrograde infusion into the pancreatic duct. Hank's salt solution is a standard salt solution mixture known in the art and available from a number of commercial sources, such as Grand Island Biological Supply Company, Grand Island, New York.

The pancreas was then removed, minced in Hank's solution at 0°C and digested with collagenase and soybean trypsin inhibitor.

All procedures were conducted at 0°C“4°C unless otherwise specified. The conditions of the digestion procedure were extremely critical. Two minced rat pancreases in an 8 ml total volume in Hank's medium were placed in a 30 ml glass tube. 2/ All glass tubes were pretreated with silicone.— The incubation mixture contained 12 mg collagenase, an enzyme prepared from Clostridium histolyticum, essentially by the method of Mandi, I., Mackennan, J.D. and Howes, E.L., J.Clin.Invest. 32, 1323 (1943), type CLS IV, obtained from Worthington Biochemical Corporation, Freehold, New Jersey, and 1 mg soybean trypsin inhibitor obtained from Sigma Chemical Company, St. Louis, Missouri. (It should be noted here that individual batches of collagenase must be pretested for optimal yield of undamaged cells, since commercial preparations vary from batch to batch and the yield of intact islets can vary considerably from one batch of enzyme to another. Thus, collagenase should be empirically selected on the basis of the yield of intact islets per rat pancreas a particular batch of collagenase is capable of giving and this should be at least 20 intact islets per rat pancreas and preferably up to 200 intact islets per rat pancrease). 4 7 27 ‘4 Incubation was carried out at 37°C for 25 minutes with shaking at the rate of 90 strokes per minute. Continuous inspection was required to insure that the collagenase digestion had proceeded to an optimal extent. If the incub5 ation was too short, the islet cells were incompletely released and if the incubation was too long the islet cells would begin to lyse. Following incubation the tube was centrifuged for 1 minute at 200 G. The supernatant was decanted and the pellet washed with Hank's solution, and this procedure was repeated five times. After the final 3/ centrifugation, the pellet was suspended in 15 ml. Ficoll,having a density of 1.085. A layer of 8 ml. Ficoll of density 1.080 was added, a layer of 5 ml. Ficoll of density 1.060 was added, and the tube was centrifuged in a swinging bucket rotor for 5 minutes at 500 G followed by 5 minutes at 2000 G. As a result of the 2/ Siliclad, Trade Mark, Clay-Adams Division; BectonDickinson Inc., Parsippany, New Jersey. 2/ Trade Mark, Pharmacia Chemical Company, Uppsala, Sweden. ί 7 2 7 ·1 foregoing process, acinar cells remained at the bottom of the tube and islet cells rose in the gradient and formed a bank between the two top layers. The islet cell band contained contaminating gaglion cells, lymph nodes, and connective tissue. Large contaminating fragments were removed from the material in the band. The remainder of the preparation was placed under a dissecting microscope where visible contaminating materials were removed by hand using a micropipette. The cell preparation was then diluted in Hank's solution and centrifuged. The supernatant was decanted and the cell pellet stored frozen in liquid nitrogen.

Islet cells pooled from 200 rats were homogenized in 4 M guanidinium fC Λ thiocyanate containing 1 Μ p-mercaptoethanol buffered to pH 5.0 at 4°C. The homogenate was layered over 1.2 ml, 5.7 M CsCl containing 100 mM EDTA and centrifuged for 18 hours at 37,000 rpm in the SW 50.1 rotor of a Beckman (Registered Trade Mark) Ultraeentrifuge at 15°C (Beckman Instrument Ccnpany, Fullerton, California). RNA traveled to the bottom of the tube.

Polyadenylated RNA was isolated by chromatography of the total RNA preparation on oligo(dT)-cellulose according to the procedure of Aviv, H., and Leder, P., supra.

Avian myeloblastosis virus reverse transcriptase, provided by D.J.

Beard, Life Science Inc., St. Petersburg, Florida, was used to transcribe total polyadenylated RNA from rat islets of Langerhans into cDNA. The reactions were carried out in 50 mM Tris-HCl, pH 8.3, 9mM MgCl^, 30 mM NaCl, 20 mM beta-mercaptoethanol, 1 mM each of 3 nonradioactive deoxyribonucleoside triphosphates, 250 juM of the fourth deoxynucleoside triphosphate labeled with specific activity 50-200 curies per mole, 20jug/ml oligo-dTj2_jg (polydeoxythymidylate preparation wherein the range of molecular lengths if frcm 12 to 18 deoxynucleotides fran Collaborative Research, Waltham, Massachusetts), 100 pg/ml polyadenylated RNA and 200 units/ml reverse transcriptase. The mixture was incubated at 45°C for fifteen minutes. After addition of EDTA-Na^ to 25 itM, the solution was extracted with an equal volume of water-saturated phenol, followed by chromatography of the aqueous phase on a Sephadex G-100 column, 0,3 cm in diameter hy 10 cm in height, in 10 nM Tris-HCl, pH 9,0, 100 rtM NaCl, 2 mM EDTA. Nucleic acid eluted in 4/ Tridom, Trade Mark, Fluka AG Chemische Fabrik, Bushs, Switzerland. - 32 47274 the void volume was precipitated with ethanol after addition of ammonium acetate, pH 6.0, to 0.25 M. The precipitate was collected by centrifugation, the pellet was dissolved te 50 jil of freshly prepared 0.1 M NaOH and incubated at 70°C for 20 minutes to hydrolyze the RNA. The mixture was 32 neutralized by the addition of 1 M sodium acetate, pH 4.5, and the P-cDNA product was precipitated with ethanol and redissolved in water. Aliquots of single stranded cDNA were analyzed on native polyacrylamide gels by the method of Dingman, C.W., and Peacock, A.C., Biochemistry 7, 659 (1968). The gels were dried and the 32P DNA detected by autoradiography 5/ using Kodak No-Screen NS-2T film. The cDNA was heterodisperse, as judged by the electrophoresis pattern. It contained at least one prominent cDNA species of about 450 nucleotides, ,as judged hy comparison with known standards.

Example 2 The synthesis and characterization of double stranded cDNA containing the sequence of rat insulin is described. The single stranded cDNA product of Example 1 was treated with reverse transcriptase to synthesize the complementary strand. Reaction mixture contained 50 mM Tris-HCl, pH 8.3, mM MgClj, 10 mM dithiothreitol, 50 mM each of three unlabeled deoxyribo32 nucleoside triphosphates, 1 mM of an alpha- P-labeled nucleoside triphosphate of specific activity 1-10 curies per millimole, 50 jig/ml cDNA and 220 units/ml of reverse transcriptase. The reaction mixture was incubated at 45°C for 120 minutes. The reaction was stopped by addition of EDTA-Na, to 25 mM, extracted with phenol and chromatographed on Sephadex G-100 followed by ethanol precipitation. An aliquot of the reaction product having 500 cpm to 1,000 cpm was analyzed hy gel electrophoresis as described in Example 1. A heterodisperse band centering around 450 nucleotides te length was observed, as determined by comparison with standard samples. Aliquots of the DNA reaction products of Example 1 and Example 2 were separately treated by digestion with an excess of restriction endonuclease Hae III and similarly analyzed by gel electrophoresis. Both the products / Trade Mark, Eastman Kodak Corporation, Rochester, New York. - 33 47 2 7 d were cleaved by the endonuclease so that two bands of radioactivity were observed on gel electrophoresis. The bands resulting from the cleavage of double stranded cDNA represented essentially the same length cleavage products as did those resulting from the cleavage of the single stranded cDNA.

Example 3 The blunt-end ligation of Hind III deeanucleotlde linkers to rat islet double stranded cDNA of Example 2 is described. The double stranded reaction product of Example 2 at a concentration of 2-5 /ig/ml was treated with 30 units of SI nuclease having an activity of 1200 units/ml, obtained from Miles Laboratories, Elkhart, Indiana, in 0.03 M sodium acetate, pH 4.6, 0.3 M sodium chloride, 4.5 mM ZnCl^ at 22OC for 30 minutes incubation followed by an additional 15 minutes incubation at 10°C. Addition of Tris-base to 0.1 M final concentration, EDTA to 25 mM, and E. coli tRNA, prepared by the method of von Ehrenstein, G., Methods in Enzymology, S.P.

Colowlck and N.O. Kaplan, Eds., Vol. 12A, p. 588 (1967), to 40jug/rnl was used to stop the digestion. After phenol extraction of the reaction mixture no and Sephadex G-100 chromatography, the 3 Ρ-cDNA eluted in the void volume was precipitated with ethanol. This treatment resulted in a high yield of cDNA molecules with base-paired ends necessary for the blunt-end ligation to chemically synthesized decanucleotides. Hind III decamers were prepared hy the method of Scheller, R.H., Dickerson, R.E., Boyer, H.W., Riggs, A.D. and Itakura, K., Science 196, 177 (1977). Ligation of Hind III decamers to cDNA was carried out by incubation at 14°C in 66 mM Tris-HCl, pH 7.6, 6.6 mM MgC^, 1 mil ATP, 10 mM dithiothreitol, 3 mM Hind III decamers having 10^ cpm/pmol and T4 DNA ligase, approximately 500 units/ml, for one hour. The reaction mixture was then heated to 65°C for 5 minutes to inactivate the ligase. KCl to 50 mM final concentration, β-mercaptoethanol to 1 mM final concentration and EDTA to 0.1 mM final concentration were added prior to digestion with 150 units/ml Hsu I or Hind III endonuclease for 2 hours at 37°C. Hind III and Hae III endonuclease are commercially available from New England Bio-Labs, Beverly, Massachusetts. The reaction - 34 47 274 product was analyzed by gel electrophoresis as in Example 1 and a peak corresponding to a sequence of approximately 450 nucleotides was observed, in addition to fragments of cleaved Hind XII decatners.

Example 4 The formation of a recombinant plasmid and its characterization after replication is described. Plasmid pMB-9 DNA, prepared as described by Rodriguez, R.L., Boliver, F., Goodman, H.M., Boyer, H.W., and Betlach, M., in ICN-DCLA Symposium on Molecular and Cellular Biology, D. P. Wierlich, W. J. Rutter, and C. F. Fox, Eds., (Academic Press, New York 1976) pp. 471-477, was cleaved at the Hind III restriction site with Hsu I endonuclease, then treated with alkaline phosphatase, type BAPF, Worthington Biochemical Corporation, Freehold, New Jersey. The enzyme was present in the reaction mixture at the level of 0.1 unit/microgram DNA and the reaction mixture was incubated in 25 mM TriS-HCl, at pH 8 for 30 minutes at 65°C, followed by phenol extraction to remove the phosphatase. After ethanol precipitation, the phosphatase treated plasmid DNA was added to cDNA containing Hind III cohesive termini at a molar ratio of 3 moles plasmid to 1 mole cDNA. The mixture was incubated in 66 mM Tris, pH 7.6, 6.6 mM MgCl2, 10 mM dithiothreitol, and 1 mM ATP for one hour at 14°C in the presence of 50 units/ml of T4 DNA ligase.

The ligation mixture was added directly to a suspension of Eh coli X-1776 cells prepared for transformation as follows: Cells were grown O to a cell density of about 2 x 10° cells/ml in 50 ml of medium containing Tryptone 10g/l, yeast extract 5 g/1, NaCl 10 g/1, NaOH 2mM, diaminopimelic acid 100 jjg/ml and thymine 40 pg/ml, at 37°C. Cells were harvested by centrifugation for 5 minutes at 5,000 G at 5°C, resuspended in 20 ml cold NaCl 10 mM, centrifuged as before and resuspended in 20 ml transformation buffer containing 75 mM CaClj, 140 mM NaCl and 10 mM Tris pH 7.5, and allowed to remain 5 minutes in ice. The cells were then centri30 fuged and resuspended in 0.5 ml transformation buffer. Transformation was carried out by mixing 100 pi of the cell suspension with 50 jul - 35 17 2 7 4 r recombinant DNA (1 pg/ml). The mixture was incubated at 0°C for 15 minutes, then transferred to 25°C for 4 minutes, then at 0°C for 30 minutes. The cells were then transferred to agar plates for growth under selection conditions.

Screening for recombinant plasmids was carried out at 5 micrograms/ml tetracycline for transformation into the Hind III site. A selected recombinant, designated pAU-1, was isolated. Crude plasmid preparations oi 2 pg - 5 pg DNA isolated from pAU-1 were digested with an excess of Hsu I endonuclease. EDTA-Na2 10 mM, and sucrose 10% w/v (i.e., weight to volume), final concentration were then added and the mixture resolved on an 8%w/v polyacrylamide gel. The DNA was found at a position corresponding to about 410 base pairs in length. In a similar experiment, plasmid pBR322 was employed as the transfer vector. All conditions were as described except final selection of recombinant clones was carried out on plates containing 20 pg/ml ampicillin.

Example 5 The DNA from pAU-1 as described in Example 4 was further purified by electrophoresis on a 6% w/v polyacrylamide gel. After elution from the gel the DNA was labeled by incubation with Y -^p-ATP and the enzyme poly20 nucleotide kinase under conditions described by Maxam and Gilbert, supra.

The enzyme catalyzes the transfer of a radioactive phosphate group from Ύ -32p_ATP to the 5'-ends of the DNA. The enzyme was obtained from E. coli by the method of Panet, A., et al., Biochemistry 12, 5045 (1973).

The DNA thus labeled was cleaved with Hae III endonuclease as described in Example 2, and the two labeled fragments, about 265 and 135 base pairs respectively, were separated on a polyacrylamide gel under the conditions described in Example 1. The isolated fragments were subjected to specific cleavage reactions and sequence analysis according to the method of Maxam and Gilbert, supra. The sequence of table 2 is based upon a composite of the findings from this series of experiments and those of a similar series of cDNA using plasmid vectors derived from col El such as pMB-9 and pBR-322.

In the sequence at the 5’ end, a sequence estimated between 50-120 - 36 4727 Λ nucleotides in length is undetermined and the poly dA segment at the 3'-end is of varying length. This sequence is provided as representing the best information presently available, with the understanding that ongoing studies may reveal additional details or may indicate a need for slight revision in some areas. The corresponding amino acid sequence of rat proinsulin I begins at the triplet position marked 1 and ends at triplet position marked 86. Some uncertainty remains with respect to the sequence underlined with a dashed line. oi CJ f—4 23 ca Eh E- *4 CJ CJ CJ <1 CJ XJ CJ Eh CJ CJ Η CJ CJ Eh CJ CJ EH CJ Eh CJ CJ < Eh O CJ CJ O τ'* Eh n Eh *4 CO CJ CJ CJ CJ CJ CJ < <4 <4 < CJ Eh CJ CJ EH CJ CJ CJ £-* Eh -< CJ CJ CJ o cj Eh CJ CJ *4 CJ CJ CJ E-* a cj CJ cj CJ a Eh a cj u o CJ Π3 OJ G Ti (U T3 c □ o *4 CJ Eh CJ e-i cj EH £h Eh O CJ o Oi Eh CJ H g CJ CJ Eh CJ CJ Eh CJ Eh CJ H CJ CJ CJ s CJ Eh CJ CJ £h CJ CJ o < »-< CJ EH a cj Eh CJ CJ *4 Eh CJ <4 CJ CJ &H o cp O CJ t CJ CJ CJ CJ £h e CJ CJ **> o a Η £h CJ < ξ CJ CJ CJ <4 CJ Eh CJ < Eh Eh CJ CJ CJ CJ CJ CJ CJ <4 CJ CJ CJ 2 CJ e CJ Η Eh < Ss CJ *4 AJ CJ CJ CJ c CJ Η Eh •H AJ c-l CJ 3 a o <4 a μ CJ r* CJ CD O o CJ Eh CJ C Eh < £h P CJ CJ CJ CJ AJ CJ CJ CJ c < £h CJ Φ CJ CJ «4 co CJ 0) CJ £h CJ μ Eh EH Eh P CJ < < CJ M-i h4 CJ CJ O <2 e CJ CJ CJ CJ ω Eh ca < &H Eh| cu CJ CJ μ CJ CJ CJI *4 CJ CJ EhI II Eh *5 *41 CJ *4 <1 co CJi C <4 CJ E~l o <4 «4 t-~>{ Ή CJ CJ ur 4J t) CJ o *4 Cl) CJ o CJ W CJ CJ Eh *a CJ CJ CJ QJ <5 CJ < μ CJ CJ co o u w CJ EH CJ < o Η CJ CJ vD CJ H CJ CJ O Η <4 <4 CJ CJ £h O CJ < EH <4 CJ CJ <4 7 2 7-1 .-- tv Example 6 A nucleotide sequence coding for human insulin is isolated, purified and incorporated in a plasmid essentially as described in Examples 1-4, starting from human pancreas tissue isolated from a suitable human source such as a donated pancreas or a fresh cadaver or a human insulinoma.

A microorganism is produced, essentially as described in Example 4, having a nucleotide sequence coding for the human insulin A chain and B chain. The known amino acid sequence of human insulin A chain is: Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile—lys-Ser-Leu-Tyr-GlU-Lsu-Glu-Asn20 Tyr-Cys-Asn The known amino acid sequence of the human insulin B chain is: Phe-Val-Asn-Glu-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val20 30 Cys-Gly-GIu-Ara-Gly-Phe-Phe-Tyr-Thr-Pro-Lys-Thr The amino acid sequences are numbered from the end having a free amino group. See Smith, L.F., Diabetes 21 (suppl. 2), 458 (1972).

Example 7 The isolation and purification of DNA having the entire structural gene sequence for RGH is described, together with the synthesis of a transfer vector containing the entire structural gene for RGH and the construction of a microorganism strain containing the gene for RGH as part of its genetic makeup.

Where genes of non-human origin are involved, the C.S. Federal safety restrictions do not require the isolation of cDNA in such a high degree of purity as that required for human cDNAs. Therefore, it was possible to isolate the cDNA containing the entire RGH structural gene by isolating electrophoretically separated DNA of the expected length, about 800 base-pairs, as determined from the known amino acid length of RGH.

Cultured rat pituitary cells, a sub-clone of the cell line GH-1, available from American Type Culture Collection, were used as a source of RGH mRNA. See Tashjian, A.H., et al., Endochrinology 82, 342 (1968). In such cells, when grown in formal conditions, growth hormone mRNA represents only a small percentage 1-3.1 w/w of the total poly-A containing SNA. However, growth hormone mRNA levels were raised above that of other cellular mRNA - 33 47271 species by che synergistic action of thyroid hormones and glucocorticoids. RNA was obtained from 5 χ 10θ cells grown in suspension culture and induced for growth hormone production by including 1 mil dexamethasone and 10 nM L-triiodothyronine in the medium for 4 days before cell collection Polyadenylated RNA was isolated from the cytoplasmic membrane fraction of the cultured cells, as described elsewhere. See Martial, J.A., Baxter, J.D., Goodman, H.M. and Seeburg, P.H., Proc.Nat.Acad.Sci. USA 74, 1816 (1977), and Bancroft, F.C., Wu, G. and Zubay, G., Proc-Nat.

Acad.Sci. USA 70, 3646 (1973). The mRNA was further purified and transcribed into double-stranded cDNA essentially as described in examples 1, 2 and 3, supra. Upon fractionation by gel electrophoresis, a faint but distinct band corresponding to a DNA of about 800 base-pairs length was observed.

Treatment of total cDNA transcribed from the cultured pituitary cell mRNA with Hhal endonuclease yielded two major DNA fragments upon electrophoretic separation corresponding to approximately 320 nucleotides (fragment A) and 240 nucleotides (fragment B). Nucleotides sequence analysis of fragments A and B as described in example 5, revealed that these fragments were in fact portions of the coding region for RGH, based on published RGH amino acid sequence data and by comparison with other known growth hormone sequences. See Wallis, M. and Davies, R.V.N., Growth Hormone And Related Peptides (Eds., Copecile, A. and Muller, E.E.), pp 1-14 (Elsevier, New York, 1976), and Dayhoff, M.O., Atlas of Protein Sequence and Structure. _5, suppl. 2, pp 120-121 (National Biomedical Research Foundation, Washington, D.C., 1976). When the 800 base-pair double-stranded cDNA isolated electrophoretically as described, supra, was similarly subjected to Hhal endonuclease treatment, two fragments corresponding in length to fragments A and B were found among the major cleavage products.

Since the approximately 800 base-pair RGH-cDNA was not purified by resort to restriction endonuclease treatment, it was necessary to treat the DNA in order to remove any unpaired single-strand ends. In practice, - 39 4-7 27 4 treatment to remove such unpaired ends was carried out prior to electrophorectic separation in 25jul of 60 mM Tris-HCl, pH 7.5, 8 mM MgCl^, mM p-mercaptoethanol, 1 mM ATP and 200 juM each of dATP, dTTP, dGTP and dCTP. 'Hie mixture was incubated with 1 unit of E. coli DMA polymerase I at 10°C for 10 minutes to exonucleolytieally remove any 3' protruding ends and to fill any 5' protruding ends. DBA polymerase I is commercially available from Boehringer-Mannheim Biochemicals, Indianapolis, Indiana.

The approximately 800 base-pair RGH-cDNA was treated by the addition of chemically synthesized Hind III linkers, as described in Example 3.

The plasmid pBR-322, having an ampicillin resistance gene and a single Hind III site located within a tetracycline resistance gene was pretreated with Hind III endonuclease and alkaline phosphatase, as described in Example 4. The treated plasmid was combined with the 800 base-pair RGH-cDNA in a DNA ligase reaction mixture as described in Example 3. The ligase reaction mixture was used to transform a suspension of Ei coli X-1776 cells, treated as previously described in Example 3. Recombinant colonies were selected by growth on nutrient plates containing ampicillin, and by inability to grow on 20 jug/ml tetracycline. Ten such colonies were obtained all of which carried plasmid with an insert of approximately 800 base-pairs that was released by Hind III cleavage.

The 800 base-pair RGH-DNA was isolated in preparative amounts from recombinant clone pRGH-1 and its nucleotide sequence determined as described in Example 5. In this instance, the nucleotide sequence included portions of the 5' untranslated region of RGH, as well as a 26 amino acid sequence found in the growth hormone precursor protein prior to secretion. The messenger of the mRNA sequence deduced from the gene sequence is shown in Table 3. The predicted amino acid sequence is in good agreement, except in positions 1 and 8, with the partial amino acid sequence of rat growth hormone as described by Wallis and Davies, supra, which comprises residues 1-43, 65-69, 108-113, 133-143 and 150-190. - 40 47274 Table 3 the The DNA nucleotide sequence of one strand, containing/entire sequence coding for RGH. Corresponding amino acids are shown, together with their position number relative to the amino terminus. Negatively numbered amino acids represent the pre-growth hormone sequence. The corresponding mRNA sequence is the same, except that U replaces T in the mRNA. □ u rt x x a ω cj o x ύ o rt X X O rt U X X p o x: cj X < a rt X X CJ □ CJ rt X X O o a p o cu o p 5£ < c CJ r-l < CD CJ C. CJ ω rt < r4 o < cd rt X l—l CJ < o O 4J CJ M 5) H I S < cd cj O CD X CD s Em CJ o Em ϋ < CJ CD cd X a I I 3 O rt CJ c CJ Cfl CJ rt J4 P. i—l < is X CJ CU Em CD o X «2 e CJ Cfl O rt CJ 3 CD r-l 5s CJ r-4 rt O CJ O Em Κ < X CJ 01 CJ rt O 3 CJ (fl •rl Λ H rt Em 5s PS O EU Em X O X «rt 3 CJ rt EM O 3 CD 3 CD rt «Μ o CO rt £m r-4 «rt X CJ < CD X O CD CJ tn CJ rt em 3 CD P X •rl r-l o rt X 5s «rt CJ < CD X CJ X X c CJ 3 CD P CD X CJ fl l-M < rt rt X CD CJ CD CJ ca χ > o rt CJ rt a rt O 00 CD r-4 rl CJ Em P CD < o < CJ fU Η rt CJ CO C H ω o cuO P o tfl <5 P O (fl < «rt CJ < < < a . < CJ □ o C CD 3 Em P CD rt Em fl ·<. rt X rt CJ X o CD O X O ca X r-l CD rt Sm 3 CD P CJ rt •—I Em rt H CJ > CD l-t < X Em. X < rt i_, P O 3 < 5s X r—i CJ rt CJ r-i X o CD ca Em CJ CD CD CD C C-, P EM AJ O rt f-t co 5s< rt Em X < Em Em < CU X rt CJ 00 o Q, CJ AJ CD r-l Ρ O V) «rt rt < o < o < O S < rt C_4 OCO P E” O 3 CD j= e-> - X CJ O rt X cu CD U Em < rM X CJ 3 CD 5s -£ bO < P CJ rt O r-l CD ♦ B CJ rt CJ >4 CJ CD .CD < < ca < P Em 3 CD c CJ C o rt CJ i4 < X < tn «rt ca < CD C CJ CJ < *rt P CJ O O c CD P CD ai o P CJ X -rt X CJ ca Em Ci CJ o o X < 3 CJ Ο Em rt o rt X rt j-t ι—ί £m X CJ χ X X £m H -rt o fU X O CJ P CJ 3 CD 0 CJ P CJ 5s < X «rt X X X CJ Em Em CD CD X < AJ CJ rt O 3 CD dOCD JU Em r-l a X «rt P CJ C < O CD CD «rt rt O tO £4 (fl CD P CD r-l CJ P CD *5 rt CD C o < O X -rt ca o EM 3 CD 5iU 3 CD P u rd X CD rt X cu CJ CJ CJ o O X CD □ <*. rt o P CJ rt X rt EM X H X O x-x >4 H Pm Em Em «rt cu X rt EM 3 CD . O 0 o c CD r-l o r-l «ίί v£3 p CJ X < CD CD CD Pm CD CD CJ XEM tn <4 rt CJ X CD «—I CD 5s <2 X CJ rt X CD CJ X -2 < CJ > CD rt P CJ o < o CJ r—l CJ P CJ P O CJ EM EM cu o Pm O 3 CD P CJ rt CJ 5^ CD r-l < X CJ X X X O CD CD Η < X -rt CD CD c CUCJ P o 3 CJ r-< <2 Ol X o rt X o CJ < CJ Em «rt X CJ Q fM rt Em 3' CD cucj P o r-4 CJ X «rt P CD CU CJ <; o o CD X X Q. CD (Q Em P -rt P «rt P CJ r4 CJ rt CJ rt p Em EM <ί o ca H ca X 0) CD 5S «ίί >4 «2 c. o ui <£ < CD ρ H 5s < X Em Si o o W CD 5s < ►4 «rt CJ P o rt a ca < SsU X CD CD CD CU O U) CD o CD rt X x a o 1-i < o a c cj h 4-1 Q rt. X fG «rt CD rt Em X a rt X r-l CJ < CJ 0 3 0 CM r—l 5s CD r-l O o cd CD 1—I «rt cd o cj o cd ai χ X CJ 3.0 « < < O - 41 □ cd r4 «ί cj cd rt «rt r-l CJ < CD CD U H x o C_ CJ ω < < o w o 5S < X < rt O r-l CJ < o cu cj w -rt < cd rt X X £m X H Em CD O U O «rt Em X cd CJ o CJ CJ cj X cj EM o X < a < CJ o «rt Em rt CJ X X rt X r-4 CJ -rt O ta x 5s O CJ X P o rt o ca «rt rt cd X CJ < CJ rt η Λ EM X X co o p cd < CJ co o P cd «rt o O 01 X 00 5s CD r-l CJ X « CD is < X *2 AJ CD rt em Du <1 r-l O rt Η > o co o P o < o □ o rt Em X O P CJ 5s -rt Em X 7 27 4 Example 8 The isolation and purification of the entire gene sequence coding for HGH is described, together with the synthesis of a recombinant plasmid containing the entire structural gene for HGH, and the production of a microorganism having the entire structural gene for HGH as part of its genetic makeup is described.

Tlie isolation of HGH mRNA is carried out essentially as described in Example 7, except that the biological source material is human pituitary tumor tissue. Five benign human pituitary tumors, quick-frozen in liquid nitrogen after surgical removal, weighing 0.4 g to 1.5 g each were thawed and homgenized in 4 M guanidinium thiocyanate containing 1 M β-mercaptoethanol buffered to pH 5.0 at 4°C. The homogenate was layered over 1.2 ml 5.7 M CsCl containing 100 mM EDTA and centrifuged for 18 hours at 37,000 rpm in the SW 50.1 rotor of a Beckman ultra-centrifuge at 15°C (Beckman Instrument Company, Fullerton, California). RNA travelled to the bottom of the tube. Further purification, using an oligo-dT column and sucrose gradient sedimentation was as described previously in Examples 1, 2 and 3. About 10% of the RNA thus isolated coded for growth hormone, as judged by incorporation of a radioactive amino acid precursor into anti-growth hormone precipitate material in a cell-free translation system derived from wheat germ. See Roberts, B.E. and Patterson, B.M., Proc.Nat.Acad.

Sci. OSA 70, 2330 (1973). Preparation of HGH-cDNA is carried out essentially as described in Example 7. The HGH-cDNA is fractionated by gel electrophoresis and material migrating to a position corresponding to about 800 nucleotides in length is selected for cloning. The selected fraction is treated with DNA polymerase I as described in Example 6, then treated by the end addition of Hind III linkers. The cDNA is then recombined witb alkaline phosphatase-treated plasmid pBR-322 using DNA ligase. E. coli X-1776 is transformed with the recombinant DNA and a strain containing HGH DNA is selected. The HGH-DNA containing strain is grown in preparative amounts, the HGH-DNA isolated therefrom and the nucleotide sequence thereof determined. The cloned HGH-DNA is found to comprise nucleotides coding for the entire amino acid sequence of HGH.

The first twenty-three amino acids of HGH are H-Phe-Pro-Tiir-Ile-Pro10 20 Leu-Ser-Arg-Leu-Phe-Asp-Asn-Ala-Met-Leu-Arg-Ala-His-Arg-Leu-His-Gln-Leu-.

The remainder of the sequence is shown in Table 4. - 42 4727 4 Table 4 Nucleotide sequence of one strand of HGH-DNA. The numbers refer to the amino acid sequence of HGH beginning at the amino terminus. The DNA sequence shown corresponds to the mRNA sequence for HGH, except that U replaces T in the mRNA. 3 O rt x P CJ xc CU CJ trt rt □ CJ rt H p ?M rt CJ >4 CJ Em X < CJ r4 CJ co H a cj 3 CJ r-t CJ □ rt 3 rt eoo rt X CO H r-l rt QJ C-t P CJ a cj U CJ > CJ CJ CJ h-3 CJ rt o P CJ CU CJ a cj 3 CJ 3 CJ rt rt in cj »—t rt rt X rt χ rd CJ C CJ CJ CJ ι-l O X u rt CJ CJ Em ω < < rt 3 rt © 3 CJ eo cj CU CJ 3 CJ rt =m o 3i < P CJ 01 rt r-l 'ϊ C CJ i-ϊ CJ rt rt < CJ CJ CJ rd *4 CJ CJ 3 CJ 3 CJ X CJ 01 c- o rM fj Ul rt w rt X e • 00 □ CJ rt rt rt Hj{ CJ CJ 75 CJ rM > a rt X 1-4 CJ P CJ rt cj AJ CJ C O □ cj rt CJ rH *J rt Em rM u-l rt a CO X rt CJ X < rt rt M rt 53 X tn rt ω cj 3 CJ rt cj en cj Xrt 45 =m rt Η ι-l P CJ P rt CU EM X o 33 CJ rt cj rt CJ CO c c r-f C? P CJ P rt 3 O l—| rt rt χ 45 CJ rt CJ rt E- P X CJ CJ > CJ Em rt CO CM l-J CJ Xrt X X c rt Ρ X C -rt 3 O rt CJ r-l < QJ CJ r-l -rt rt rt 45 Em « CJ CJ CJ co <; CJ CJ rt rt Ph SM >> rt X rt P rt en cj rt cj p rt P rt 45 CJ P CJ r-l X c a X < <; < H rt Em < Em < <—! □ rt 3 CJ o XCJ CU CJ 3 e rd rt rt χ cm X © co < rd rt □ rt e a hJ cj CJ CJ rt a O CJ 3 z3 3 CJ rt cj a < rt cj r—I CJ r-l *?. X < 05 £m « CJ CJ CJ CU X O CJ Ph £m > CJ S' 5 ω cj 3 CJ 3 CJ rt CJ tn cj S- CJ X < rd < o rt < < CJ CJ CJ CJ >-3 rt ►4 rt P CJ cu a 3 CJ X © 3 -rt ρ a cuo rt x rt Em w rt rt cj > CJ X CJ co < rt CJ r-ι X W < p a ο CJ (X CJ b 9 o co rt o P CJ 03 < Xrt rt P* p x CO X pu a rt CJ Η Em X < x < g-ι χ O CJ 3 CJ W CJ P CJ PUCJ P CJ r-t rt X< 45 CJ ω rt rt a r—Η CJ P-. CJ CJ CJ X < Η <; rt cj rt o P rt 3 CJ3 3 O cn CJ O 45 CJ rt Em rt Em rd rt Xrt □ rt r-l < tO X < kJ a iJ CJ CJ o bJ < eno CJ CJ O CJ CU CJ 3 CJ O OT CJ P CJ P CJ rt Em sr xrt P CJ3 -5 rd rt CJ CJ P-ι CJ Em X >-l CJ r—1 u rt rt rt rt x P CJ pu CJ rt cj rt o r-l X rt cj w -rt 45 E-i X Em rt X H < CO X <} o PU Em Pi EM 45 X P-. X p X 3 CJ P EM rt CJ W CJ rt o X rt X< rd Em XCJ 3 CJ r-l rt CO X CJ CJ EM Em l-d rt O EM P CJ CJ CJ 3 CJ QJ CJ rd CJ 3 CJ r-l rt X Em rt Em rd rt Xrt 3 CJ CJ CJ H rt > CJ CJ O Em Η r—1 rt cj a P rt 3 CJ 3 a XCJ 3 CJ rt CJ rt H tn < rd CJ QJ Em p a CO Em i-J CJ C -3 CJ CJ U CJ xrt £-i X rt CJ 3 CJ P CJ p Em 3 O 45 X rt X rt o 45 CJ rt X P CJ fU X jJ CJ CO -rt EM rt r-3 O 45 CJ X < cn χ 3 CJ CU CJ eo cj XCJ X CJ o rt X « -rt P CJ rd CJ CU CJ a X oo X CJ .-rt CJ rt o CJ o W rt 3 CJ P cj ‘Ρ H o a O P CJ rt X rt o rt a P CJ to xrt Q) c-i X CJ CO EM CO &M {U CJ mM Η X 45 X 0-. X P CJ rt cj rt a P CJ 3 CJ rt o r-l Em rd o rt CJ 52 *S rt o CO X M < rt o co <; rt rt —I o rt CJ P CJ CO CJ xo XCJ (0 CJ Λ CJ P CJ rd CJ rd CJ X rt ►4 rt CJ X rt rt a CJ CJ CJ CJ o a ϋ cj C-l CJ cj X cj CJ X CJ rt cj cj CJ o rt o X a X CJ.

CJ CJ X CJ cj o X cj co < X a) CJ 45 X X X XCJ rd CJ a cj ω X XCJ a x p a ω u ω < XO rH CJ CJ CJ 3 CJ r—I -¼ CJ CJ rd CJ rt X > o - 43 4 7 2? 4 With the method of the present invention as illustrated above it has become possible for the first time to isolate a nucleotide sequence coding for a specific regulatory protein from a higher organism such as a vertebrate, and transfer the genetic information contained therein to a microorganism where it may be replicated indefinitely.

The disclosed method may be applied to the isolation and purification of the human insulin gene, and to its transfer to a microorganism. Similarly, the human growth hormone gene, and other protein genes may be isolated, transferred to and replicated in a microorganism. Several novel recombinant plasmids are disclosed, each containing within its nucleotide sequence a subsequence having the structure of and transcribed from a gene of a higher organism.

Several novel microorganisms are disclosed, each modified to contain a nucleotide sequence having the structure of and transcribed from a gene of a higher organism. The practice of the invention has been illustrated by demonstrating the transfer of the rat gene for the proinsulin I to a strain of Escherichia coli, and to the transfer of the rat gene for growth hormone to a strain of E. coli. The sequence of the main portion of the transferred gene has been determined in each case and has been found to code for the entire amino acid sequence of rat proinsulin X, or rat growth hormone, respectively as determined by reference to the known genetic code which is common to all forms of life.

Claims (56)

1. A DNA transfer vector containing within its nucleotide sequence a subsequence having the structure of and transcribed from a gene of a higher organism (as herein described). 5

2. A DNA transfer vector according to claim 1 wherein the gene of a higher organism codes for insulin (us herein described).

3. A DNA transfer vector according to claim 1 comprising a nucleotide sequence coding for the A chain of human insulin comprising: 5’---GGL^Ti^GTL^AJ^AJ^GKJG^ACL^RgSgATM^TGK^QR^S^ X 13 TY 13 TAK i4 CAJ 15 X l6 TY 16 GAJ 17^ K 18 TAK 19 TGK 20 AAK 21— 3 ’ wh&rein A is deoxyadenyl, G is deoxyguanyl, C is deoxycytosyl, T is thymidyl, J is A or G; K is ΐ or G; L is A, T,C or G; M is A, C or T; X n is T or C, if Y n is A or G, and C if Y n is c or T; Y n is A, G, C or T, if X n is C , and A or G if X n is T; W is C or A, if z is G or A, and C if Z is C or T; n n n Z n is A, G, C or T, if is C , and A or G if W n is A; QR is TC, if S is A, G, C or T, and AG if S is T or C; η η n S n is A, G, C or T, if QR q is TC, and T or C if QR& is AG 25 and subscript numerals, n, refer to the amino acid position in human insulin, for which the nucleotide sequence corresponds, according to the genetic code, the amino acid positions being numbered from the amino end.

4. A DNA transfer vector according to claim 3 comprising an additional nucleotide sequence coding for the B chain of human insulin 30 comprising: - 45 7 a 7 -ι

5. '---TTK 1 GTL 2 AAK 3 GAJ 4 CAK 5 X 6 TY g TGK 7 GGL 8 QR 9 S 9 CAK 10 X 11 TY 11 GTL 12 GAJ ]J GCL H X 15 TY w TAK 1 gX 17 TY 17 GTL 18 TGK 1 gGGL 20 GAJ 21 W 22 GZ 22 GGL 23 ^24^25^26^27^28^29^30-^5. A DNA transfer vector according to claim 4 wherein the nucleotide sequences coding for the A and B chains of human insulin are joined in the partial sequence 5'---ACL 3Q GCL^---3' and wherein from 1 to 100 nucleotide triplets of sequence (N N,N ), wherein Na, Nb and Nc may be A, T, G or C a d c may be interposed in continuous sequence between said ACL 3Q and GCL^ provided that NaNbNc is neither TAJ nor TGA.

6. The DNA transfer vector of claim 3 transferred to and replicated in a microorganism strain.

7. The DNA transfer vector of claim 6 wherein the microorganism is a bacterium and the DNA transfer vector is a plasmid.

8. The DNA transfer vector of claim 7 wherein the bacterium is selected from Escherichia coli X-1776 and Escherichia coli RR-1 and the plasmid is selected from pMB-9 and pBR-322.

9. The DNA transfer vector of claim 4 transferred to and replicated in a microorganism strain.

10. The DNA transfer vector of claim 9 wherein the microorganism is a bacterium and the DNA transfer vector is a plasmid.

11. The DNA transfer vector of claim 10 wherein the bacterium is selected from Escherichia coll X-1776 and Escherichia coli RR-1 and the plasmid is selected from pMB-9 and pBR-322.

12. A DNA transfer vector of claim 5 transferred to and replicated in a microorganism strain.

13. The DNA transfer vector of claim 12 wherein the microorganism is a bacterium and the DNA transfer vector is a plasmid. 4 7 3''-!

14. The DNA transfer vector of claim 13 wherein the bacterium is selected from Escherichia coli X-1776 and Escherichia coli RR-1 and the plasmid is selected From pMB-9 and pBR-322. 5 15. The DNA transfer vector of claim 2 comprising a nucleotide sequence coding for the amino acid sequence of rat proinsulin I, said sequence comprising: 5'---ITT GTC AAA CAG CAC CTT TGT GGT CCT CAC CTG GTG GAG GCT CTG TAC CTG GTG TGT GGG GAA CGT GGT TTC TTC TAC ACA CCC AAC TCC CGT CGT GAA GTG GAG 10 GAC CCG CAA GTG CCA CAA CTG GAG CTG GCT GGA GGC CCG GAG GCG GGG GAT CTT CAG ACC TGG GCA CTG GAG GTT GCC CGG CAG AAG CGT GGC ATT GTG GAT CAG TGC TGC ACC AGC ATC TGC TCC CTC TAC CAA CTG GAG AA.C TAC TGC AAC- —3' wherein A is deoxyadenyl, G is deoxyguanyl, C is deoxycytosyl, and T is thymidyl. 16. The DNA transfer vector of claim 15 comprising plasmid pBR-322 transferred to and replicated in Escherichia coli RR-1. 17. A DNA transfer vector according to claim 1 wherein the gene 20 of a higher organism (as herein described) codes for growth hormone. 18. A DNA transfer vector according to claim 17 comprising a nucleotide sequence coding for the amino acid sequence of human growth hormone, said sequence comprising: 5''TTK 2 CCL2ACL3ATH/ 1 CGL 5 X 6 TY 6 QRyS 7 H g GZgX g TYgTTK 10 GAK 11 AAK 1 2 G 14CL- L3 ATGX 15 Ty^ 5 25 ^16^16^^17^^18^19^^19^20^20^^21^^22^23 TX 23 GCL 24 TTK 25 CAK2 6 ACL2 7 TAK 23 CAJ 2g CAJ 30 TTK 31 CAJ 32 CA.J 3r \CL 34 Tx\K 35 ATM 36 CCL3 7 AxV 3 gG f \J 3g C.\J, 0 .UJ 41 TAK 42 qR 43 S 43 TTK 44 X 45 TY 45 CAJ 46; V\K 47 CCL Z(S Cx\J 49 ACL 5 oQR 5 iS 51 X5 2 TY 52 TGK 53 TTK 54 QR5 5 S 53 GA J 56qR5 7 S 57 ATN5 8 CCL 59A c t6o c CL61qR f . 2 S 6? AiXK 63 H 64 GZ 64 GAJ 65 CAJ 66 ACL 67 CAJ 68 CAJ 69 iUJ 70 QR 71 S 71 AAK 72 X 73 TY 73 CiU 74 X 75 TY 75 X 76 T ’’7& W 77 GZ 77 Ara 78^79 S 79 X 80 TY 80 X 81' rY Sl^ 2 ^ S2 Ara 83 C.U S4 QR S5 S S5 TG %6 XTg 7 GA J88 CCL 89 GTL 90 CAJ gl TIK g2 X g3 TY 93 H g4 GZ g4 QR g5 S g5 GTl, g6 TTK 97 GCL gs AAK gg .UK 100 X 101 TY 101 - 47 •5 7 2 7 4 GTL 102 T -' lK i03 GGL 104 GCL l05 GR 106 S 10S GAK 107 QR lo3 S 108 AjVK 109 GTL 110 TAK iii GlXK n2 X 113 TY 113 X 114 TY n4‘ UJ 115 GAK 116 X l 17^117^113^119^120^121^122^123 X 124 TY i24 ATG 125 GGL 126 W 127 GZ 127 X 123 TY 128® J 129 GAK 13O GGL 131 CR 132 S 132 CCL 133 • i 134 G2 134 ACL 135 GCL 136 CAJ 137 Ar ‘ I 133 TTX 139 AAJ 140 CAJ 141 ACIj 142™143 Q ’ A 144 S 144 ^14 5 ^Μ6 ϋΛΚ 147 Λε 448 Α ^149^150 3 130^15Γ' ΑΚ 152 ί?Λί '153 ΰΛΚ ΐ54 ί:(:Ι -155 :< 156 ΊΎ 136 .TY X,..TY,,.TAK.,,TGK,,„ΤΤΚ,,.W,,.GZ, AAJ χ 157 ΤΥ 1 57 ^158 ίΑΑΚ 159 ΤΑΙί 160 εε 461·Ί62 1 ^62\63 Χ ^63' 1 ^164 τ ^65' ΙΤΚ 166 ν 'ΐό7 υΛ 167 168 GAK l69 ATC 17O GAK 171 AA;r i72 CTIj 173® J 174 aa '175 ,n:K 176 X 177 lrY 177 W 178 GZ 178 a ' IM 179 GTL 180 C;U 181 TGK 182 w 183 GZ lS3 c ! R i84 S 184 GTL 185 CAJ 186 GGL lS7 QR 188 S lSS TGK 189 GGL 190 TTK 19: - 3' wherein A is deoxyadenyl, G is deoxyguanyl, C is deoxycytosyl, T is thymidyl, J is A or G; K is I or C; L is A, T,C or G; :i is A, C or T; is T or C, if Y is Λ or G, and C if Y is C or T; n n Y n is A, , G, C or T, if x n is C, and A or G if X is T n w n is C or A, if Z n is G or A, and C if Z n is G or T; 2 n is A, , C, C or T if Wn is G, and A or G if W. is A n QR is TC, if S n is A, G, C or T, and AG if S n is T or C; S n is A, G, C or T, if QR n is TC, and T or C if Q?. n is AG and subscript numerals, n, refer to the amino acid position in human growth hormone, for which the nucleotide sequence corresponds, according to the genetic code, the amino acid positions being numbered from the amino end. 19. The transfer vector of claim 18 transferred to and replicated in a microorganism. 20. The transfer vector of claim 19 wherein the transfer vector is selected from pMB-9 and pBR-322 and the microorganism is a bacterium selected from Escherichia coli X-1776 and Escherichia coll RR-1. 48 4 7 27 4 21. A method of making a DNA transfer vector having a nucleotide sequence coding for insulin comprising: (a) isolating islet cells containing mRNA coding for insulin from the pancreas of an insulin-producing organism, 5 (b) extracting the mRNA from the islet cells in the presence of an RNase inhibitor composition, whereby RNase degradation of the mRNA is prevented, (c) separating the mRNA substantially free of protein, DNA and other RNA, 10 (d) synthesizing a double stranded cDNA wherein one strand has a nucleotide sequence complementary to that of the mRNA, thereby producing cDNA having a nucleotide sequence coding for insulin, (e) providing a DNA transfer vector having reactant ends capable of being joined together or of being joined with double stranded cDNA,and

15. (f) joining the DNA transfer vector with the double stranded cDNA having a nucleotide sequence coding for insulin, whereby a DNA transfer vector having a nucleotide sequence coding for insulin is produced. 22. A method for isolating islet cells containing mRNA enriched with respect to the nucleotide sequence coding for insulin according to claim

16. 20 21 comprising: treating pancreas tissue containing islet cells and other cells with a hydrolytic enzyme and a trypsin inhibitor to free the islets from the other cells, fractionating the treated tissue in order to obtain islets 25 essentially free from other cells and cell debris, isolating mRNA from the islets, said RNA being enriched with respect to nucleotide sequence coding for insulin. - 49 23. A method according to claim 22 wherein the hydrolytic enzyme is collagenase which is empirically selected as hereinbefore defined.

17. 24. Λ method according to claim 22 wherein the hydrolytic enzyme treatment is carried out in silicone-treated glassware.

18. 25. A method according to claim 22 wherein the hydrolytic enzyme treatment is carried out in plastic ware.

19. 26. The method of claim 21 wherein the RNase inhibitor composition comprises a chaotropic cation, a chaotropic anion and a disulfide bond breaking agent.

20. 27. The method of claim 26 wherein the chaotropic cation is guanidinium and the chaotropic anion is thiocyanate.

21. 28. The method of claim 26 wherein the disulfide bond breaking agent is j3-mercaptoethanol. 29 · The method of claim 21 wherein the RNase inhibitor composition comprises 4 M guanidinium thiocyanate and 0.2 M )3-mercaptoethanol.

22. 30. The method of claim 21 wherein the DNA molecules having reactant ends are joined in step (f) by a method whereby a selected portion of the reactant ends are prevented from joining to each other comprising: pretreating a selected portion of the reactant ends with a reagent capable of removing the 5' terminal phosphate groups therefrom, incubating the DNA molecules having pretreated and untreated reactant ends together with a DNA ligase enzyme, whereby a joining reaction is catalyzed between said reactant ends, except that the pretreated reactant ends are not joined to each other hy the ligase-catalyzed reaction.

23. 31. A method according to claim 30 wherein the reagent for pretreating the reactant ends is alkaline phosphatase.

24. 32. A method according to claim 30 wherein the DNA molecules to be joined comprise a mixture of restriction endonuclease treated plasmid DNA and non-plasmid linear DNA, and Che portion selected for pretreatment comprises the plasmid DNA, whereby end-to-end joining of the plasmid DNA in the absence of recombination uith the non-plasmid DNA, is prevented

25. 33. A method of making a DNA transfer vector having a nucleotide sequence coding for growth hormone comprising: (a) isolating pituitary cells containing mRNA coding for growth hormone, (b) extracting the mRNA from the pituitary cells in the presence of an RNase inhibitor composition, whereby RNase degradation of the mRNA is prevented, (c) separating the mRNA substantially free of protein, DNA and other RNA, (d) synthesizing a double stranded cDNA wherein one strand has a nucleotide sequence complementary to that of the mRNA, thereby producing cDNA having a nucleotide sequence coding for growth hormone, (e) providing a DNA transfer vector having reactant ends capable of being joined together or of being joined with double stranded cDNA, and (f) joining the DNA transfer vector with the double stranded cDNA having a nucleotide sequence coding for growth hormone, whereby a DNA transfer vector having a nucleotj.de sequence coding for growth hormone is produced.

26. 34. The method of claim 33 wherein the RNase inhibitor composition comprises a chaotropic cation, a chaotropic anion and a disulfide bond breaking agent.

27. 35. The method of claim 34 wherein the chaotropic cation is guanidinium and the chaotropic anion is thiocyanate.

28. 36. The method of claim 34 wherein the disulfide bond breaking agent is jS-mercaptoethanol.

29. 37. The method of claim 33 wherein the RNase inhibitor composition comprises 4 M guanidinium thiocyanate and 0.2 M S-mercaptoethanol. 1 7 2 7 1

30. 38. The method of claim 33 wherein the DNA molecules having reactant ends are joined in step (f) by a method whereby a selected portion of the reactant ends are prevented from joining to each other comprising: pretreating a selected portion of the reactant ends with a reagent capable of removing the 5' terminal phosphate groups therefrom, incubating the DNA molecules having pretreated and untreated reactant ends together with a DNA ligase enzyme, whereby a joining reaction is catalyzed between said reactant ends, except that the pretreated reactant ends are not joined to each other by the ligase-catalyzed reaction.

31. 39. A method according to claim 38 wherein the reagent for pretreating the reactant ends is alkaline phosphatase.

32. 40. A method according to claim 38 wherein the DNA molecules to he joined comprise a mixture of restriction endonuclease treated plasmid DNA and non-plasmid linear DNA, and the portion selected for pretreatment comprises the plasmid DNA, whereby end-to-end joining of the plasmid DNA in the absence of recombination with the non-plasmid DNA, is prevented.

33. 41. A method for making a microorganism having a nucleotide sequence coding for insulin comprising: (a) isolating islet cells containing mKNA coding for insulin from the pancreas of an insulin-producing organism, (b) extracting the mRNA from the islet cells in the presence of an RNase inhibitor composition, whereby RNase degradation of the mRNA is prevented, (c) separating the mRNA substantially free of protein, DNA and other RNA, (d) synthesizing a double stranded cDNA wherein one strand has a nucleotide sequence complementary to that of the mRNA, thereby producing cDNA having a nucleotide sequence coding for insulin, (e) providing a DNA transfer vector having reactant ends capable of being joined together or of being joined with double stranded cDNA, (f) joining the DNA transfer vector with the double stranded cDNA having a nucleotide sequence coding for insulin, and (g) mixing a microorganism together with the DNA transfer vector joined with cDNA having a nucleotide sequence coding for insulin, whereby 5 a microorganism having a nucleotide sequence coding for insulin is produced

34. 4 2 . A method for isolating islet cells containing mRNA enriched with respect to the nucleotide sequence coding for insulin according to claim 41 comprising: treating pancreas tissue containing islet cells and other cells 10 with a hydrolytic enzyme and a trypsin inhibitor to free the islets from the other cells, fractionating the treated tissue in order to obtain islets essentially free from other cells and cell debris, isolating mRNA from the islets, said RNA being enriched with respect 15 to nucleotide sequence coding for insulin.

35. 43. A method according to claim 42 wherein the hydrolytic enzyme is collagenase which is empirically selected as hereinbefore defined.

36. 44. A method according to claim 42 wherein the hydrolytic enzyme treatment is carried out in silicone-treated glassware. 20

37. 45. A method according to claim 42 wherein the hydrolytic enzyme treatment is carried out in plastic ware.

38. 46. The method of claim 41 wherein the RNase inhibitor composition comprises a chaotropic cation, a chaotropic anion and a disulfide bond breaking agent. 25

39. 47. The method of claim 46 wherein the chaotropic cation is guanidinium and the chaotropic anion is thiocyanate.

40. 48. The method of claim 46 wherein the disulfide bond breaking agent is S-mercaptoethanol. - 53

41. 49. The method of claim 41 wherein the RNase inhibitor composition comprises 4 M guanidinium thiocyanate and 0.2 Ϊ-I )3-mercaptoetfianol.

42. 50. The method of claim 41 wherein the DNA molecules having reactant ends are joined in step (f) by a method whereby a selected portion of the reactant ends are prevented from joining to each other comprising: pretreating a selected portion of the reactant ends with a reagent capable of removing the 5' terminal phosphate groups therefrom, incubating the DNA molecules having pretreated and untreated reactant ends together with a DNA ligase enzyme, whereby a joining reaction is catalyzed between said reactant ends, except that the pretreated reactant ends are not joined to each other by the ligase-catalyzed reaction.

43. 51. A method according to claim 50 wherein the reagent for pretreating the reactant ends is alkaline phosphatase.

44. 52. A method according to claim 50. wherein the DNA molecules to be joined comprise a mixture of restriction endonuclease treated plasmid DNA and non-plasmid linear DNA, and the portion selected for pretreatment comprises the plasmid DNA, whereby end-to-end joining of the plasmid DNA in the absence of recombination with the non-plasmid DNA, is prevented.

45. 53. A method for making a microorganism having a nucleotide sequence coding for growth hormone comprising: (a) isolating pituitary cells containing mRNA coding for growth hormone, (b) extracting the mRNA from the pituitary cells in the presence of an RNase inhibitor composition, whereby RNase degradation of the mRNA is prevented, (c) separating the mRNA substantially free of protein, DNA and other RNA, (d) synthesizing a double stranded cDNA wherein one strand has a nucleotide sequence complementary to that of the mRNA, thereby producing cDNA having a nucleotide sequence coding for growth hormone, (e) providing a DNA transfer vector having reactant ends capable of being joined together or of being joined with double stranded cDNA, (f) joining the DNA transfer vector with the double stranded cDNA having a nucleotide sequence coding for growth hormone, and 5 (g) mixing a microorganism together with the DNA transfer vector joined with cDNA having a nucleotide sequence coding for growth hormone, whereby a microorganism having a nucleotide sequence coding for growth hormone is produced.

46. 54. The method of claim 53 wherein the RNase inhibitor composition 10 comprises a chaotropic cation, a chaotropic anion and a disulfide bond breaking agent.

47. 55. The method of claim 54 wherein the chaotropic cation is guanidiniua and the chaotropic anion is thiocyanate.

48. 56. The method of claim 54 wherein the disulfide bond disrupting 15 agent is ^-mercaptoethanol.

49. 57. The method of claim 53 wherein the RNase inhibitor composition comprises 4 M guanidinium thiocyanate and 0.2 M y8-mercaptoethanol.

50. 58. The method of claim 53 wherein the DNA molecules having reactant ends are joined in step (f) by a method whereby a selected portion of the 20 reactant ends are prevented from joining to each other comprising: pretreating a selected portion of the reactant ends with a reagent capable of removing the 5' terminal phosphate groups therefrom, incubating the DNA molecules having pretreated and untreated reactant ends together with a DNA ligase enzyme, whereby a joining reaction is 25 catalyzed between said reactant ends, except that the pretreated reactant ends are not joined to each other by the ligase-catalyzed reaction.

51. 59. A method according to claim 58 wherein the reagent for pretreating the reactant ends is alkaline phosphatase. - 55

52. 60.. A method according to claim 58 wherein the DNA molecules to be joined comprise a mixture of restriction endonuclease treated plasmid DKA and non-plasmid linear DNA, and the portion selected for pretreatment comprises the plasmid DNA, whereby end-to-end joining of the plasmid DNA in the absence of recombination with the non-plasmid DNA, is prevented.

53. 61.. A method of making a DNA transfer vector having a nucleotide sequence coding for a gene of a higher organism (as herein described) comprising: (a) isolating cells containing mRNA coding for the desired protein, (b) extracting the mRNA from the cells, if necessary in the presence of an RNase inhibitor to prevent RNase degradation of the mRNA, (c) separating the mRNA substantially free of protein, DNA and other RNA, (d) synthesizing a double stranded cDNA wherein one strand has a nucleotide sequence complementary to that of the mRNA, thereby producing cDNA having a nucleotide sequence coding for the desired protein, (e) providing a DNA transfer vector having reactant ends capable of being joined together or of being joined with double stranded DNA, and (f) joining the DNA transfer vector with the double stranded cDNA having a nucleotide sequence coding for the desired protein, whereby a DNA transfer vector having a nucleotide sequence coding for the desired protein is produced.

54. 62. A DNA transfer vector according to claim 1, substantially as described in any one of Examples 3 to 8

55. 63. A method according to claim 21 or 33 of making a DNA transfer vector substantially as described 5 in Examples 1 to 3, or any one of Examples 4 to 8.

56. 64. A method according to claim 41 or 53 for making a microorganism substantially as described in any one of Examples 4 to 8.