GB1565190A - Recombinant dna transfer vector and microorganism containing a gene from a higher organism - Google Patents

Abstract

DNA transfer vectors which contain in their nucleotide sequence a subsequence which has the structure of the gene of a higher organism and which has been produced by a transcription on a gene of the higher organism are described. For example, this gene can encode the A chain or the B chain of human insulin or else be a gene which encodes the growth hormone of a higher organism. The DNA transfer vector is prepared by isolating from cells an mRNA which encodes the desired protein, specifically essentially free of proteins, DNA and other RNA. Then a double-stranded cDNA is synthesised, in which one strand has a nucleotide sequence which is complementary to that of the mRNA, in this way a cDNA is obtained which has a nucleotide sequence which encodes the desired protein. A DNA transfer vector with reactive ends is linked to the double-stranded cDNA, resulting in the desired DNA transfer vector. The DNA transfer vectors can be used to modify microorganisms which then contain a nucleotide sequence having the structure of a gene of a higher organism.

Description

(54) RECOMBINANT DNA TRANSFER VECTOR AND MICROORGANISM CONTAINING A GENE FROM A HIGHER ORGANISM (71) We, THE REGENTS OF THE UNIVERSITY OF CALIFORNIA, an educational mstitution chartered by the State of California, United States of America, having a principal place of business at 2200 University Avenue, City of Berkeley, State of California, RJ.S.A., do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed to be particularly described in and by the following statement: The present invention relates to the isolation of a specific nucleotide sequence which contains the genetic information coding for a specific protein, the synthesis of DNA having this specific nucleotide sequence and transfer of that DNA to a microorganism host wherein the DNA may be replicated. More specifically, the present invention relates to the isolation of the insulin gene and the growth hormone gene, their purification, transfer and replication in a microbial host and their subsequent characterization. Novel products are produced according to the present invention. These products include a recombinant plasmid containing the specific nucleotide sequences derived from a higher organism and a novel microorganism containing as part of its genetic makeup a specific nucleotide sequence derived from a higher organism.

The symbols and abbreviations used herein are set forth in the following table: DNA - deoxyribonucleic acid A - Adenine RNA - ribonucleic acid T - Thymine cDNA - complementary DNA G - Guanine (enzymatically synthesized C - Cytosine from an mRNA sequence) Tris - 2-Amino-2-hydroxy mRNA - messenger RNA ethyl-l ,3-propanediol tRNA - transfer RNA EDTA - ethylenediamine dATP - deoxyadenosine triphosphate tetraacetic acid dGTP - deoxyguanosine triphosphate ATP - adenosine triphosphate dCTP - deoxycytidine triphosphate TTP - thymidine triphosphate The biological significance of the base sequence of DNA, is as a repository of genetic information. It is known that the sequence of bases in DNA is used as a code specifying the 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.

The 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 RNA. 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 RNA are capable of entering into the same kind of base pairing relationships that exist with DNA.

Consequently, the RNA transcript of a DNA nucleotide sequence will be complementary to the sequence copied. Such RNA i s 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 by the translational process into a functional protein. An example is provided in the case of insulin.

GENETIC CODE Phenylalanine(Phe) TTK Histidine(His) CAK Leucine(Leu) XTY Glutamine(Gln) CAJ Isoleucine(Ile) ATM Asparagine(Asn) AAK Methionine(Met) ATG Lysine(Lys) AAJ Valine(Val) GTL Aspartic acid(Asp GAK Serine(Ser) ORS Glutamic acid(Glu) CAJ Proline(Pro) CCL Cysteine(Cys) TGK Threonine(Thr) ACL Tryptophan(Try) TGG Alanine(Ala) GCL Arginine(Arg) WGZ Tyrosine(Tyr) TAK Glycine(Gly) GGL Termination signal TAJ Termination signal TGA Key: Each 3-letter triplet represents a trinucleotide of DNA, having a 5' end on the left and a 3' end on the right. The letters stand for the 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 T 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 is W is C Z = A or G if W is A QR = TC if S is A, G, C or T QR = AG if S is T or 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 T 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.Natl.Acad.Sci.

USA 73, 1964 (1976) and Lomedico, P.T. and Saunders, G.F., Nucl.Acids Res. 3, 381 (1976). The structure of the preproinsulin molecule can be represented schematically as H-(pre-peptide)-B chain-(C peptide)-A chain-OH.

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 been 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 the gene coding for a protein of medical significance, from an organism which normally makes the protein to an appropriate microorganism. In this way, the protein can be mae 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 processed after synthesis. Furthermore, isolated genetic sequences may 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 oligo-deoxynucleotide 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 complementary 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. 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 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 oligo-nucleotides bearing the restriction site sequence. Therefore virtually any segment of DNA can be coupled to any other segment simply by attaching the appropriate restriction oligonucleotide to the ends of the molecule, and subjecting the product 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).

S1 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 ligase is capable of catalyzing blunt end ligation in which two molecules having blunt ends are covalently joined. See Sgaramella, V., Van de Sande, J.H., and Khorana, H.G., Proc.Natl.Acad.Sci. USA 67, 1468 (1970).

Alkaline phosphatase is an enzyme of general specificity capable of hydrolyzing phosphate esters including 5' terminal phosphate 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 108 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 rate insulin gene is described in detail. Insuline was chosen for this effort because of its central significance from the standpoint of clinical medicine, and from the standpoint of basis research. The disclosed procedure is applicable by those of ordinary skill in the are to the isolation of the insulin gene of other organisms, including humans.

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. The 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, e.g., the possibility of establishing symbiotic relationships between microorganisms produced pursuant to this invention and human beings with chronic or acute deficiency diseases, whereby microorganisms genetically altered as herein 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 nuceotide sequence coding for the desired protein, whereby a DNA transfer vector having a nucleotide sequence coding for the desired protein is producted.

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: 5'---GGL1 ATM2 GTL3 GAJ4 CAJ5 TGK6 TGK, ACL8 QRg Sg ATMlo TGK11 QR12 X13 TY13 TAK14 GAJ15 X16 TY16 GAJ17 AAK18 TAK19 TGK20 AAK21 ---3 Such a DNA transfer vector may comprise an additional nucleotide sequence coding for the B chain of human insulin comprising: 5'---TTKl GTL2 AAK3 GAJ4 CAK5 X6 TY6 TGK7 GGL8 QRg Sg CAK10 X11 TYl, GTLl2 GAJ13 GCL,4 Xs TY15 TAK16 X17 TY17 GTL18 TGK19 GGL20 GAJ21 W22 GZ22 GGL23 TTK24 TTK25 TAK 6 ACL27 CCL28 AAJ29 ACL30---3' Also, the nucleotide sequence coding for the A and B chains of human insulin may be joined in the partial sequence 5'---ACL3(, GCL---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 ACL3(, and GCL1 provided that NaNbNc 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 the 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, 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, (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 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.

Thus, there are provided methods for isolating a specific nucleotide sequence containing genetic information, synethesis 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 rate 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 method. 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 m 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 selfcomplementary 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 be 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 nucleotlde 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, transferred 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 soucre, including human 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 c 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 for the purpose of transferring an intact genetic sequence to a micro-organism. 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 somtimes 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 if 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 operatons 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 undegrated mRNA in good yield from isolated islets of Langerhans of rat pancreas. of 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, and diiodosalicylate. The relative effectiveness of salts formed by combining such cations and anions will be determined in part by their solubility. For example, 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 guanidinuim thiocyanate.

Thiol compounds, such as P-mercaptoethanol are known to break intra-molecular disulfide bonds in proteins by a thiol-disulfide interchange reaction. Many thiol compounds are known to be effective, including besides P-mercaptoethanol, dithiothreitol, cysteine, propanol, and dimercaptan. Aqueous solubility in a necessary property, since the thiol compound must be present in large excess over the intra-molecular disulfides, in order to drive the interchange reaction essentially to completion, ss-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 relatonship 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 is much greater, hence it can be 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 foward 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 denatuant 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 rive 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 p-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 M 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.

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. In 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 be selectively isolated by chromatography on columns of celluslose to which is attached oligo-thymidylate, as decribed by 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 drawing 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 complementary to the purified mRNA. The enzyme of choice for this reaction is reverse 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. It is convenient to procide that one of the deoxynucleoside triphosphates be labelled with 32P in the alpha positon 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.

As shown 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 on Sephadex (Trade Mark, Pharmacia Inc., Uppsala, Sweden.) 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. the 32P labeled cDNA may be concentrated by ethanol precipitation is 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 a-32P labeled nucleoside triphosphate. Reverse transcriptase is available from 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, itmay be convenient to purify the DNA from the reaction mixture. As described previously, it has been found convenient to employ the steps of phenol extraction, chromatography on Sephadex G-100 and ehtanol 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 S1 nuclease isolated from Aspergillus oryzae. The enzyme may be purchased from Miles Research Products, Elkhart, Indiana. Treatment of the hairpin DNA structue with S1 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 S1 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 DNA 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 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). The enzyme, Hae III, from Hemophilus 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 the same recognition site, as Hind III. These two enzymes are therefore considered as functionally interchangeable.

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 1. 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 by 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 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 bacteriophage 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, O., 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., ICN-UCLA Symposium on Molecular Mechanisms In The Control of Gene Expression, D.P. Nierlich, W.J. Rutter, C.F. Fox, Eds. (Academic Press, NY, 1976), pp.

471-477. Plasmids derived from col El are characterized by being relatively small, having molecular weights of the order of a few millions 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 by 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 additon to the tetracycline resistance gene, a gene for amplicillin resistance. The presence of the drug resitance genes provides a convenient way for selecting cells which have been successfully infected by the plasmid, since colonies of such cells with 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 but for purposes of public safety, narrowly restricted. 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. Alicrobiol., 30, 507 (1976). E. coli RR-1 is suitable where P3 containment facilities are available. As in the case of the lasmids, 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 endonuclease-treated plasmid DNA with cDNA containing end groups similarly treated. In order to minimize the change 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 device DNA vectors having a restriction endonuclease site m 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 the 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+4.

The present invention is based on the fact that the DNA ligase catalyzed reaction takes place between a 5'-phosphate DNA end group 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

Case . Reactants Lipase Product I 3' 5' 3' 5' OH H2O3OP O-P-O + +2H2O OPO3H2 HO O-P-O 5' 3' 5' 3' II 3' 5' 3' 5' OH H2O3P-O O-P-O OH OH OH HO 5' 3' 5' 3' III 3' 5' OH + HO no reaction OH + HO 5' 3' In Table 1, double-stranded DNA is schematically represented by solid parallel lines, while their respective 5' and 3' end groups are labeled with hydroxyl (OH) or phosphate (OPO3H2), as the case may be. In case I. 5' phosphates occur on both reactant ends with result that both strands become covalently joined. In case II only one of the strands to be 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 be 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.

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 E. coli may be used, Modrich, P., and Lehman, I.R., J.Biol.chem. 245, 3626 (1970).

The efficiency of reconstituting the original sequence from sub-fragments 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, end 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 II, 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.

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 selcted 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 by Hind III or HSU I endonuclease digestion and were subjected to DNA sequence analysis bg 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 E. coli. A nucleotide sequence approximately 800 nucleotides in length was reisolated after extensive replication in E. coli 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.

Example 1 The described procedures demonstrate the extraction and isolation of rat insulin 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 00C 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 critic 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 4M guanidinium thiocyanate (Tridom, Trade Mark, Fluka AG Chemische Fabrik, Buchs, Switzerland.) containing 1M ,8-mercaptoethanol buffered to pH 5.0 at 4"C. The homogenate was layered over 1.2 ml, 5.7 M CsC1 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) Ultracentrifuge at 15"C (Beckman Instrument Company, 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 rate islets of Langerhans into cDNA. The reactions were carried out in 50 mM Tris-HC1, pH 8.3, 9mM MgC12, 30 mM NaC1, 20 mM beta-mercaptoethanol, 1 mM each of 3 nonradioactive deoxyribonucleoside triphosphates, 250 M of the fourth deoxynucleoside triphosphate labeled with o:-32p, specific activity 50-200 curies per mole, 20 Fg/ml oligo-dT12 l8 (polydeoxythymidylate preparation wherein the range of molecular lengths if from 12 to 18 deoxynudeotides from Collaborative Research, Waltham, Massachusetts), 100 Fg/ml polyadenylated RNA and 200 units/ml reverse transcriptase. The mixture was incubated at 45"C for fifteen minutes. After addition of EDTA-Na2 to 25 mM, 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 by 10 cm in height, in 10 mM Tris-HCl, pH 9,0, 100 mM NaC1, 2 mM EDTA. Nucleic acid eluted in 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 in 50 Fi of freshly prepared 0.1 M NaOH and incubated at 70"C for 20 minutes to hyrolyze the RNA. The mixture was neutralized by the addition of 1 M sodium acetate, pH 4.5, and the 32P-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 Peakcock, A.C., Biochemistry 7 659 (1968). The gels were dried and the 2PDNA detected by autoradiography using Kodak No-Screen NS-2T (Trade Mark, Eastman Kodak Corporation. Rochester, New York.) 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 by 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 I was treated with reverse transcriptase to synthesize the complementary strand. Reaction mixture containing 50 mM Tris-HC1, pH 8.3. 9mM MgCl2, 10 mM dithiothreitol, 50 mM each of three unlabeled deoxyribonucleoside triphosphates, 1 mM of an alpha-32P-labeled nucleoside triphosphate of specific activity 1-10 curies per millimole, 50 Fg/ml cDNA and 220 units/ml of reverse transcriptase. The reaction mixture was incubated at 450C for 120 minutes. The reaction was stopped by addition of EDTA-Na2 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 by gel electrophoresis as described in Example 1. A heterodisperse band centering around 450 nucleotides in 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 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 decanucleotide 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 Fg/ml was treated with 30 units of S1 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 ZnC11 at 22"C 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. Colowick and N.O. Kaplan, Eds., Vol. 12A, p. 588 (1967), to 40Fglml was used to stop the digestion. After phenol extraction of the reaction mixture and Sephadex G-100 chromatography, the 32P-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 by 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-HC1, pH 7.6, 6.6 mM MgCl2, 1 mM ATP, 10 mM dithiothreitol, 3 mM Hind III decamers having 105 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.

KCI to 50 mM final concentration, ss-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 370C. Hind III and Hae III endonuclease are commercially available from New England Bio-Labs, Beverly, Massachusetts. The reaction 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 III decamers.

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-UCLA 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, for 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 140C in the presence of 50 units/ml of T4 DNA ligase.

The ligation mixture was added directly to a suspension of E. coli X-1776 cells prepared for transformation as follows: Cells were grown to a cell density of about 2 x 108 cells/ml in 50 ml of medium containing Tryptone 10g/l, yeast extract 5 g/l, NaCI 10 g/l, NaOH 2mM, diaminopimelic acid 100 llg/ml and thymine 40 llg/ml, at 37"C. Cells were harvested by centrifugation for 5 minutes at 5,000 G at 5"C, resuspended in 20 ml cold NaCI 10 mM, centrifuged as before and resuspended in 20 ml transformation buffer containing 75 mM Cacti2, 140 mM NaCI and 10 mM Tris pH 7.5, and allowed to remain 5 minutes in ice. The cells were then centrifuged and resuspended in 0.5 ml transformation buffer. Transformation was carried out by mixing 100 Ill of the cell suspension with 50 Ill recombinant DNA (1 Zg/ml). the mixture was incubated at 0 C for 15 minutes, then transferred to 25"C for 4 minutes, then at 00C 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 of 2 Rg - 5 Rg 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 pBR-322 was employed as the transfer vector. All conditions were as described except final selection of recombinant clones was carried out on plates containing 20 llg/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 labelled by incubation with Y-32P-ATP and the enzyme polynucleotide kinase under conditions described by Maxam and Gilbert, supra. The enzyme catalyzes the transfer of a radioactive phosphate group from Y-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 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.

TABLE 2 1 [undetermined]----- GCC CTG CTC GTC CTC TGG GAG CCC AAG CCT GCT CAG GCT TTT GTC AAA CAG CAC CTT TGT 10 20 30 GGT CCT CAC CTG GTC GAG GCT CTG TAC CTG GTG TGT GGG GAA CGT GGT TTC TTC TAC ACA CCC AAC TCC CGT CGT 40 50 GAA GTG GAG GAC CCG CAA GTG CCA CAA CTG GAG CTG GCT GGA GGC CCG GAG GCC GGG GAT CTT CAG ACC TGG CCA 60 70 80 CTG GAG GTT GCC CGG CAG AAG CGT GGC ATT GTG CAT CAG TGC TGC ACC AGC TC TGG TCC CTC TAC CAA CTG CAG 86 AAC TAC TGC AAC TGA GTTCAATCAATTCCCGATCCACCCCTCTGCAATGAATAAAGCCTTTGAATGAGC-poly A Scored Sections = Areas of Present Uncertainty 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: G l y-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr-Glu-Leu- 20 Glu-Asn-Tyr-Cys -Asn The known amino acid sequence of the human insulin B chain is: 1 10 Phe-Val-Asn-Glu-His-Leu-Cys-Gly-Ser-H i s-Leu-Val-Olu-Ala-Leu-Tyr- 20 30 Leu-Val-Cys-G l y-Glu-Arg-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 U.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% w/w of the total poly-A containing RNA. However, growth hormone mRNA levels were raised above that of other cellular mRNA species by the synergistic action of thyroid hormones and glucocorticoids.

RNA was obtained from 5 x 10 cells grown in suspension culture and induced for growth hormone production by including 1 mM 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 HhaI 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 HhaI 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 ncessary to treat the DNA in order to remove any unpaired single-strand ends. In practice, treatment to remove such unpaired ends was carried out prior to electrophorectic separation in 25 Rl of 60 mM Tris-HCl, pH 7.5, 8 mM MgC12, 10 mM 13-mercaptoethanol, 1 mM ATP and 200 M each of dATP, dTTP, dGTP and dCTP. The mixture was incubated with 1 unit of E. coli DNA polymerase I at 100C for 10 minutes to exonucleolytically remove any 3' protruding ends and to fill any 5' protruding ends. DNA 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 resitance 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 E. 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 pg/ml tetracycline. Ten such colonies were obtained all of which carried plasmid with an insert of approximately 800 base-pairs that was relased by Hind II 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 Davis, supra, which comprises residues 1-43, 65-69, 108-113 133-143 and 150-190.

Table 3 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 Tin the mRNA.

TABLE 3 -26 Met Ala Ala Asp Ser Gin Thr Pro Trp Leu Leu Thr Phe Ser Leu Leu Cys Leu Leu 5'---GTGGACAGATCACTGAGTGGCG ATG CCT GCA GAC TCT CAG ACT CCC TGG CTC CTG ACC TTC AGC CTG CTC TGC CTG CTG 1 20 Trp Pro Gln Glu Ala Gly Ala Leu Pro Ala Met Pro Leu Ser Ser Leu Phe Ala Asn Ala Val Leu Agr Ala Gln His Leu His Gln Leu TGG CCT CAA GAG GCT GGT GCT TTA CCT GCC ATG CCC TTG TCC AGT CUG TTT CCC AAT GCT GTG CTC CGA CCC CAG CAC CTG CAC CAG CTG 40 Ala Ala Asp Thr Tyr Lys Glu Phe Glu Arg Ala Tyr Ile Pro Glu Gly Gln Arg Tyr Ser Ile Gln Asn Ala Gln Ala Ala Phe Cys Phe GCT GCT GAC ACC TAC AAA GAG TIC GAG CGT GCC TAC ATT CCC GAG GGA CAG CGC TAT TCC ATT CAG AAT GCC CAG GCT GCT TTC TCC TTC 60 80 Ser Glu Thr Ile Pro Ala Pro Thr Gly Lys Glu Glu Ala Gln Gln Arg Thr Asp Met Glu Leu Leu Arg Phe Ser Leu Leu Leu Ile Gln TCA GAG ACC ATC CCA GCC CCC ACC GGC AAG GAG GAG GCC CAG CAG AGA ACT GAC ATG GAA TTG CTT GCG TTC TCG CTG CTG CTC ATC CAG 100 Ser Trp Leu Gly Pro Val Gln Phe Leu Ser Arg Ile Phe Thr Asn Ser Leu Met Phe Gly Thr Ser Asp Arg Val Tyr Glu Lys Leu Lys TCA TGC CTG GGG CCC GTG CAG TTT CTC AGC AGG ATC TTT ACC AAC AGC CTG ATG TTT GGT ACC TCG GAC CGC GTC TAT GAG AAA CTG AAG 120 140 Asp Leu Glu Glu Gly Ile Gln Ala Leu Met Gln Glu Leu Glu Asp Gly Ser Pro Arg Ile Gly Gln Ile Leu Lys Gin Thr Tyr Asp Lys GAC CTG GAA GAG GCC ATC GAG GCT CTG ATG CAG GAG CTG GAA GAC GGC AGC CCC CGT ATI GGG CAG ATC CTC AAG CAA ACC TAT GAC AAG 160 Phe Asp Ala Asn Met Arg Ser Asp Asp Ala Leu Leu Lys Asn Tyr Gly Leu Leu Ser Cys Phe Lys Lys Asp Leu His Lys Ala Glu Thr TTT GAC GCC AAC ATG CGC AGC GAT GAC GCT CTG CTC AAA AAC TAT GGG CTG CTC TCC TGC TIC AAG AAG GAC CTG CAC AAG GCA GAG ACC 180 Tyr Leu Arg Val Met Lys Cys Arg ARG Phe Ala Glu Ser Ser Cys Ala Phe TAC CTG CGG GTC ATG AAG TGI CGC CGC TTT GCG GAA AGC AGC TGT GCT TTC TAG GCACACACTGGTGTCTCTGCGGCACTCCCCCGTTACCCCCCTGTACT CTGGCAACTGCCACCCCTACACTTTGTCCTAATAAAA@@AA@@A@@@A@CA@ATA @@@@ A@ @ 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.

The 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 frmercaptoethanol 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 centrifued for 18 hours at 37,000 rpm in the SW 50.1 rotor of a Beckman ultra-centrifuge at 150C (Beckman Instrument Company, Fulleton, 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 o 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 . USA 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 with alkaline phosphatase-treated plasmid pBR-322 using DNA ligase. E. cli 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 10 H-Phe-Pro-Thr-Ile-Pro-Leu-Ser-Arg-Leu-Phe-Asp-Asn-Ala-Met-leu- 20 Arg-Ala-His-Arg-Leu-His-Gln-Leu- .

The remainder of the sequence is shown in Table 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.

TABLE 4 24 32 40 43 Arg Th Arg Thr Tyr Gln Glu Phe Glu Glu A@@ @@@ Ile Pro Tyr Glu Gln Tys Tyr Ser Phe Leu Gln Asn Pro Gln 3 GCC III GAC ACC TAC CAG GAG ITI GAA GAA GCC TAT AT@ CCA AAG GAA CAG AAG TAT TCA TTC CTG CAG AAC CCC CAG 60 Thr Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser Asn Asg Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu Leu Leu ACC TCC CTC TGT TTC TCA GAG TCT ATT CCG ACA CCC TCC AAC AGG GAG GAA ACA CAA CAG AAA TCC AAC CTA CAG CTG CTC 80 100 Arg Ile Ser Leu Leu Leu Ile Gln Ser Trp Leu Glu Pro Val Gln Phe Leu Arg Ser Val Phe Ala Asn Asn Leu Val Tyr CGC ATC TC@ CTG CTG CTC AT@ CAG TCG TGG CTG GAG CCC GTG CAG CTC CTC AGG AGT CTC TTC GCC AAC AAC CTG CTG TAC 120 Gly Ala Ser Asp Ser Asn Val Tyr Asp Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly Arg Leu Glu Asp GGC GCC TCT GAC AGC AAC GTC TAT GAC CTC CTA AAG GAC CTA GAG GAA GGC ATC CAA ACG CTG ATG GGG AGG CTG GAA GAC 140 Gly Ser Pro Arg Thr Gly Glu Ile Phe @ys Gln Thr Tyr Ser Lys Phe Asp Thr Asn Ser His Asn His Asp Ala Leu Leu GGC AGC CCC CGG ACT GGG CAG ATC TTC AAG CAG ACC TAC AGC AAG TTC GAC ACA AAC TCA CAC AAC CAT CAC GCA CTA CTC 160 180 Lys Asn Tyr Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr Phe Leu Arg Ile Val Gln Cys Arg Ser AAG AAC TAC GGG CTG CTC TAC TGC TTC AGG AAG GAC ATG GAC AAG GTC GAG ACA TTC CTG CGC ATC GTG CAG TGC CGC TCT 191 Val Glu Gly Ser Cys Gly Phe GTG GAG GGC AGC TGT GGC TTC TAG CTGCCCGGGTGGCATCCCTGTGACCCCTCCCCAGTGCCTCTCCTGGCC-3 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 lasmids 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 I, or rat growth hormone, respectively as determined by reference to the known genetic code which is common to all forms of life.

Claims (80)

WHAT WE CLAIM IS:

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.

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

3. A DNA transfer vector according to claim 1 comprising a nucleotide sequence coding for the A chain of human insulin comprising: 5'---GGL1 ATM2 GTL3 GAJ4 CAJ5 TGK6 TGK, ACL8 QRg S9 ATMlo TGK11 QR12 S12 X13 TY13 TAK14 GAJ15 X16 TY16 GAJ17 AAKls TAK19 TGK20 AAK21---3' wherein A is deoxyadenyl, G is deoxyguanyl, C is deoxycytosyl, T is thymidyl, J is A or G; K is T or C; L is A, T, C or G; M is A, C or T; Xn is T or C, if Yn is A or G, and C if Yn is C or T: Yn is A, G, c or T, if Xn is C, and A or G if Xn is T; Wn is C or A, if Zn is G or A, and C if Zn is C or T: Zn is A, G, C or T, if Wn is C, and A or G if Wn is A; QRn is TC, if Sn is A, G, C or T, and AG if Sn is T or C; Sn is A, G, C or T, if QRn is TC, and T or C if QRn is AG and 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 comprising: 5'---TTK1 GTL2 AAK3 GAJ4 CAK5 X6 TY6 TGK7 GGL8 QR9 Sg CAK,(, X11 TYI1 GTL12 GAJ13 GCL14 Xl5 TY15 TAK16 Xt7 TY17 GTL17 TGK19 GGL20 GAJ21 W22 GZ22 GGL23 TTK24 TTK25 TAK26 ACL27 CCL28 AAJ29 ACLgo---3'

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'---ACL30 GCLt---3' and wherein from 1 to 100 nucleotide triplets of sequence (NaNbNC), wherein Na, Nb and Nc may be A, T, G or C, may be interposed in continuous sequence between said ACL30 and GCL1 provided that N"NbNC 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 coli 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.

14. The DNA transfer vector of claim 13 wherein the bacterium is selected from Escherichia coli X-1776 and Escheichia coli RR-1 and the plasmid is selected from pMB-9 and pBR-322.

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'---TTT 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 GAC CCG CAA GTG CCA CAA CTG GAG CTG GCT GGA GGC CCG GAG GCC 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 AAC 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 of a higher organism 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'-TTK1 CCL2 ACL3 ATM4 CCL5 X6 TY6 QR7 S7 W8 GZ8 X9 TTK10 GAK11 AAK12 GCL13 ATG14 X15 TY15 W16 GZ16 GCL17 CAK18 W19 GZ19 X20 TY20 CAK21 CAJ22 X23 TY23 GCL24 TTK25 GAK26 ACL27 TAK28 CAJ29 GAJ30 TTK31 GAJ32 GAJ33 ACL34 TAK35 ATM36 CCL37 AAJ38 GAJ40 AAJ41 TAK42 QR43 S43 TTK44 X45 TY45 CAJ46 AAK47 CCL48 CAJ49 ACL50 QR51 S51 X52 TY52 TGK53 TTK53 TTK54 QR55 S55 GAJ56 QR57 S57 ATM58 CCL59 ACL60 CCL61 QR62 S62 AAK63 W64 GZ64 GAJ66 GAJ66 ACL67 CAJ68 CAJ69 AAJ70 QR71 S71 AAK72 X73 TY73 GAJ74 X75 TY75 X76 TY76W77 GZ77 ATM78 QR79 S79 X80 TY80 X81 TY81 X82 TY82 ATM83 CAJ84 QR8s S85 TGG86 X87 TY87 GAJ88 CCL89 GTL90 CAJ91 TTK92 X93 TY93 W94 GZ94 QR95 S95 GTL96 TTK97 GCL98 AAK99 AAK100 X101 TY101 GTL102 TAK103 GGL104 GCL105 QR106 S106 GAK107 QR108 S108 AAK109 GTL 110 TAK111 GAK112 X113 TY113 X114 TY114 AAJ115 GAK116 X117 TY117 GAJ118 GAJ119 GGL120 ATM121 CAJ122 ACL123 X124 TY124 ATG125 GGL126 W127 GZ127 X128 TY128 GAJ129 GAK130 GGL131 QR132 S132 CCL133 W134 GZ134 ACL135 GGL136 CAJ137 ATM138 T T K 1 3 9 AAJ140 CAJ141 ACL142 TAK143 QR144 S144 AAJ145 TTK146 GAK147 ACL148 AAK149 QR1so Siso CAK151 AAK152 CAK153 GAK154 GCL155 X156 TY156 X157 TY157 AAJ158 AAK159 TAK160 GGL161 X162 X162 TY162 X163 TY163 TAK164 TGK165 TTK166 W167 GZ167 AAJ168 GAK169 ATG170 GAK171 AAK172 GTL172 GTL,73 GAJ174 ACL175 TTK176 X177 TY177 W178 GZ178 ATM179 GTL180 CAJ181 TGK182 W183 GZ183 QR184 S184 GTL185 GAJ186 GGL187 QRl88 S188 TGK189 GGL190 TTK191 - 3' wherein A is deoxyadenyl, G is deoxyguanyl, C is deoxycytosyl, T is thymidyl, J is A or G; K is T or C; L is A, T, C or G; M is A, C or T: Xn is T or C, if Yn is A or G. and C if Yn is C or T; Yn is A, G. C or T. if Xn is C, and A or G if Xn is T; Wn is C or A, if Zn is G or A, and C if Zn is C or T; Zn is A. G. C or T. if Wn is C, and A or G if Wn is A: QRn is TC, if Sn is A. G. C or T, and AG if Sn is T or C; Sn is A. G, C or T. if QRn is TC, and T or C if QRn is AG and 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 coli RR-1.

21. A microorganism modified to contain a nucleotide sequence having the structure of, and transcribed from, a gene of a higher organism.

22. A microorganism according to claim 21 wherein the gene of a higher organism codes for insulin.

23. A microorganism according to claim 21 wherein the gene of a higher organism codes for human insulin.

24. A microorganism modified to contain a nucleotide sequence coding for the A chain of human insulin comprising: 5'---GGL1 ATM2 GTL3 GAJ4 CAJ5 TGK6 TGK7 ACL8 QRg Sg ATMlo TGK11 QR12 S12 X13 TY13 TAK14 GAJ15 X16 TY16 GAJ17 AAK18 TAK19 TGK20 AAK21---3' wherein A is deoxyadenyl, G is deoxyguanyl, C is deoxycytosyl, T is thymidyl, J is A or G; K is T or C; L is A, T, C or M is A, C or T; Xn is T or C, if Yn is A or G, and C if Yn is C or T; Yn is A, G, C or T, if Xn is C, and A or G if Xn is T; Wn is C or A, if Zn is G or A, and C if Zn is C or Zn is A, G, C or T, if Wn is C, and A or G if Wn is A; QRn is TC, if Sn is A, G, C or T, and AG if Sn is T or C; Sn is A, G, C or T, if QRn is TC, and T or C if QRn is AG and 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.

25. A microorganism according to claim 24 modified to an additional nucleotide sequence coding for the B chain of human insulin, comprising: 5'---TTK1 GTL2 AAK3 GAJ4 CAK5 X6 TY6 TGK7 GGL8 QR9 Sg CAK10 X11 TY11 GTL12 GAJ13 GCL14 X15 TY15 TAK16 X17 TYt7 GTL18 TGK19 GGL20 GAJ21 W22 GZ22 GGL23 TTK24 TTK25 TAK26 ACL27 CCL28 AAJ29 ACL30---3'.

26. A microorganism according to claim 25 wherein the nucleotide sequences coding for the A and B chains of human insulin are joined in the partial sequence 5'---ACL30 GCL1---3' and wherein from 1 to 100 nucleotide triplets of sequence (NaNbNC), wherein Na, Nb and Nc may be A, T, G or C, may be interposed in continuous sequence between said ACL30 and GCL1 provided that NaNbNC is neither TAJ nor TGA.

27. The microorganism of claim 24 wherein the nucleotide sequence coding for the A chain of human insulin is carried on a DNA transfer vector.

28. The microorganism of claim 27 wherein the DNA transfer vector is a plasmid selected from pMB-9 and pBR-322 and the microorganism is Escherichia coli.

29. The microorganism of claim 25 wherein the nucleotide sequence coding for the B chain of human insulin is carried on a DNA transfer vector.

30. The microorganism of claim 29 wherein the DNA transfer vector is a plasmid selected from pMB-9 and pBR-322 and the microorganism is Escherichia coli.

31. The microorganism of claim 26 wherein the nucleotide sequences are carried on a DNA transfer vector.

32. The microorganism of claim 31 wherein the DNA transfer vector is a plasmid selected from pMB-9 and pBR-322 and the microorganism is Escherichia coli.

33. A microorganism according to claim 21 wherein the gene of a higher organism codes for growth hormone.

34. A microorganism according to claim 21 wherein the gene of a higher organism codes for human growth hormone.

35. A microorganism modified to contain a nucleotide sequence coding for the amino acid sequence of human growth hormone, said sequence comprising: 5'-TTK1 CCL2 ACL3 ATM4 CCL5 X6 TY6 QR7 S7 W8 GZ8 X9 TTK10 GAK11 AAK12 GCL13 ATG14 X15 TY15 W16 GZ16 GCL17 CAK18 W19 GZ19 X20 TY20 CAK21 CAJ22 X23 TY23 GCL24 TTK25 GAK26 ACL27 TAK.8 CAJ29 GAJ30 TTK31 GAJ32 GAJ33 ACL34 TAK35 ATM36 CCL37 AAJ38 GAJ40 AAJ41 TAK42 QR43 S43 TTK44 X45 TY45 CAJ46 AAK47 CCL48 CAJ49 ACL50 QR51 S51 X52 TY52 TGK53 TTK54 QR55 S55 GAJ56 QR57 S57 ATM58 CCL59 ACL60 CCL61 QR62 S62 AAK63 W64 GZ64 GAJ66 GAJ66 ACL67 CAJ68 CAJ69 AAJ70 QR71 S71 AAK72 X73 TY73 GAJ74 X75 TY75 X76 TY76W77 GZ77 ATM78 QR79 S79 X80 X81 TY81 X82 TY82 ATM83 CAJ84 QR85 S85 S85 TGG86 X87 TY87 GAJ88 CCL89 GTL90 CAJ91 TTK92 X93 TY93 W94 GZ94 QR95 S95 GTL96 TTK96 TTK97 GCL98 AAK99 AAK100 X101 TY101 GTL102 TAK103 GGL104 GCL105 QR106 S106 GAK106 QR108 S108 AAK109 GTL 110 TAK111 GAK112 X113 TY113 X114 TY114 AAJ115 GAK116 X117 TY117 GAJ118 GAJ119 GGL120 ATM121 CAJ122 ACL123 X124 TY124 ATG125 GGL126 W127 GZ127 X128 TY128 GAJ129 GAK130 GGL131 QR132 S132 CCL133 W134 GZ134 ACL135 GGL136 CAJ137 ATM138 TTK139 AAJ140 CAJ141 ACL142 TAK143 QR144 S144 AAJ145 TTK146 GAK147 ACL148 AAK149 QR150 S150 CAK151 AAK152 CAK153 GAK154 GCL155 X156 TY156 X157 TY157 AAJ158 AAK159 TAK160 GGL161 X162 TY162 X163 TY163 TAK164 TGK165 TTK166 W167 GZ167 AAJ168 GAK169 ATG170 GAK171 AAK172 GTL172 GTL173 GAJ174 ACL175 TTK176 X177 TY177 W178 GZ178 ATM179 GTL180 CAJ181 TGK182 W183 GZ183 QR184 S184 GTL185 GAJ186 GGL187 QR188 S188 TGK189 GGL190 TTK191 TAGCTGCCCGGGTGGCATCCCTGTGACCCCTCCCCAGTGCCTCTCCTGGCC 3 wherein A is deoxyadenyl, G is deoxyguanyl, C is deoxycytosyl T is thymidyl, J is A or G; K is T or C; L is A, T, C or G; M is A, C or T; X, is T or C, if Yt, is A or G, and C if Yt, is C or T; Yn is A, G, C or T, if Xn is C, and A or G if Xn is T; Wn is C or A, if Zn is G or A, and C if Zn is C or T; Zn is A, G, C or T, if Wn is C, and A or G if Wn is A; QR, is TC, if S, is A, G, C or T, and AG if S, is T or C; S, is A, G, C or T, if QR, is TC, and T or C if QR, 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.

36. 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, (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. 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.

37. A method for isolating islet cells containing mRNA enriched with respect to the nucleotide sequence coding for insulin according to claim 36 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 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.

38. A method according to claim 37 wherein the hydrolytic enzyme is collagenase which is empirically selected as hereinbefore defined.

39. A method according to claim 37 wherein the hydrolytic enzyme treatment is carried out in silicone-treated glassware.

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

41. The method of claim 36 wherein the RNase inhibitor composition comprises a chaotropic cation. a chaotropic anion and a disulfide bond breaking agent.

42. The method of claim 41 wherein the chaotropic cation is guanidinium and the chaotropic anion is thiocyanate.

43. The method of claim 41 wherein the disulfide bond breaking agent is - mercaptoethanol.

44. The method of claim 36 wherein the RNase inhibitor composition comprises 4 M guanidinium thiocyanate and 0.2 M (3-mercaptoethanol.

45. The method of claim 36 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.

46. A method according to claim 45 wherein the reagent for pre-treating the reactant ends is alkaline phosphatase.

47. A method according to claim 45 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.

48. A method of making a DNA transfer vector having a nucleotide sequence coding for growth hormone comprising: a isolating pituitary cells cntaining 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 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 nucleotide sequence coding for growth hormone is produced.

49. The method of claim 48 wherein the RNase inhibitor composition comprises a chaotropic cation, a chaotropic anion and a disulfide bond breaking agent.

50. The method of claim 49 wherein the chaotropic cation is guanidinium and the chaotropic anion is thiocyanate.

51. The method of claim 49 wherein the disulfide bond breaking agent is - mercaptoethanol.

52. The method of claim 48 wherein the RNase inhibitor composition comprises 4 M guanidinium thiocyanate and 0.2 M (3-mercaptoethanol.

53. The method of claim 48 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.

54. A method according to claim 53 wherein the reagent for pretreating the reactant ends is alkaline phosphatase.

55. A method according to claim 53 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.

56. A method for making a microorganism 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 preveitted. c separating the mRNA substantially free of protein, DNA .md other RNA, (d) synthesizing a double stranded cDNA wherein one strand has ( 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 a microorganism having a nucleotide sequence coding for insulin is produced.

57. A method for isolating islet cells containing mRNA enriched with respect to the nucleotide sequence coding for insulin according to claim 56 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 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.

58. A method according to claim 57 wherein the hydrolytic enzyme is collagenase which is empirically selected as hereinbefore defined.

59. A method according to claim 57 wherein the hydrolytic enzyme treatment is carried out in silicone-treated glassware.

60. A method according to claim 57 wherein the hydrolytic enzyme treatment is carried out in plastic ware.

61. The method of claim 56 wherein the RNase inhibitor composition comprises a chaotropic cation, a chaotropic anion and a disulfide bond breaking agent.

62. The method of claim 61 wherein the chaotropic cation is guanidinium and the chaotropic anion is thiocyanate.

6J. The method of claim 61 wherein the disulfide bond breaking agent is - mercaptoethanol.

64. The method of claim 56 wherein the RNase inhibitor composition comprises 4 M guanidinium thiocyanate and 0.2 M -mercaptoethanol.

65. The method of claim 56 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.

66. A method according to claim 65 wherein the reagent for pretreating the reactant ends is alkaline phosphatase.

67. A method according to claim 65 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.

68. 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 (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.

69. The method of claim 68 wherein the RNase inhibitor composition comprises a chaotropic cation. a chaotropic anion and a disulfide bond breaking agent.

70. The method of claim 69 wherein the chaotropic cation is guanidinium and the chaotropic anion is thiocyanate.

71. The method of claim 69 wherein the disulfide bond disrupting agent is - mercaptoethanol..

72. The method of claim 68 wherein the RNase inhibitor composition comprises 4 M guanidinium thiocyanate and 0.2 M B-mercaptoethanol.

73. The method of claim 68 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-catlyzed reaction.

74. A method according to claim 73 wherein the reagent for pretreating the reactant ends is alkaline phosphatase.

75. A method according to claim 73 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.

76. A method of making a DNA transfer vector having a nucleotide sequence coding for a gene of a higher organism comprising: (a isolating cells contaifning 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.

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

78. A micro-organism according to claim 21 substantially as described in any one of Examples 4 to 8.

79. A method according to claim 36 or 48 of making a DNA transfer vector substantially as described in Examples 1 to 3, or any one of Examples 4 to 8.

80. A method according to claim 56 or 68 for making a microorganism substantially as described in any one of Examples 4 to 8.