Recombinant dna transfer vector and microogranism containig a gene from a higher organism

This invention relates to a method for producing a recombinant DNA molecule containing a nucleotide sequence encoding insulin and used in the manufacture of an i nsulin- producing microorganism.

The following symbols and abbreviations are used in this specification: DNA - deoxyribonucleic acid

RNA - ribonucleic acid cDNA - complementary DNA (enzymatically synthesized from the mRNA sequence) mRNA - messenger RNA tRNA - transfer RNA dATP - deoxyadenosine triphosphate dGTP - deoxyguanosine triphosphate dCTP - deoxycytide

A - adenine

T - tyrnine

G - guanine

C - cytosine

Tris-2-amino-2-hydroxyethyl-1,3-propanediol

EDTA - ethylenediaminetetraacetic acid

ATP - adenosine triphosphate

TTP - thymidine triphosphate

Hind III recognition site - sequence A ^ AGCTT, specific_sest cleaved with Hind ΙΙΙ the arrow-through Hsu I endonuclease recognition site - SEQ ID IaGCTT A, ti specifically cleaved by the arrow Hsu I endonuclease means.

HaeIII recognition site - SEQ ID GG ICC, specifically a broken arrow mark Search ΙΙΙ endonucleases.

Col E1 - colicicin E1-forming plasmid poly dA - polydeoxyadenylate oligo ^ 12-18 ° lig ° de ° xythymidylate (12-18 bases)

Alu I recognition site - SEQ ID AG, CT, specifically a broken arrow mark Alu I endonuclease means.

Bam HI recognition site - SEQ G | GATCC, specifically a broken arrow Bam HI - endonucleases.

BglII-recognition site - SEQ ID GCCNNNN ^ NGGC, specifically a broken arrow into the BglII endonucleases (Note: N stands for nucleotides).

Eco RI recognition site - SEQ ID ^ G AATTC, specifically a broken arrow into the Eco RI endonuclease means.

Eco RII recognition site - SEQ ID JrCCAGG or IcCTGG, specifically a broken arrow into the Eco RLI endonucleases.

Search 11-point identification sequence - AGCGC iT, AGCGC 1 C,

GGCGC or GGCGCi G specifically cleaved by means of the arrow Search II endonuclease.

Hinc II identification site - sequence GTTAAC, GTTGAC,

GTCAAG or GTCGAC, specifically cleaved by Hinc II endonuclease.

4 6 4 0

Pst I recognition site - SEQ ID CTGCA G ^, specifically a broken arrow into the Pst I endonucleases.

Sal I recognition site - SEQ G ^ TCGAC, specific cleavage of the arrow into the Sal I endonucleases. The Sst I - the recognition site - SEQ ID GAGCT C ^, specifically a broken arrow into the Sst I-endonucleaasin means.

The biological significance of the base sequence of DNA lies in the fact that it is a repository of genetic information. It is known that the base sequence of DNA is the code by which the amino acid sequence of all proteins produced by a cell is determined. In addition, portions of the sequence may have the function of regulating the time and amount of protein production. The nature of the regulatory units is incompletely known. The base sequence of each strand is used as a template when DNA is replicated in cell division.

The way in which DNA base sequence information is used to determine the amino acid sequence of proteins is essentially the same for all living organisms.

It has been shown that each amino acid normally present in proteins is determined by one or more trinucleotides, i.e., triplet sequences. Thus, for each protein, there is a corresponding DNA segment containing the triplet sequence corresponding to the amino acid sequence of the protein. The genetic code is shown in the following table.

<td rowspan="2">Fenyy lialan iini.</td><td colspan="4">Genetic code</td>

<td>(Phe)</td><td>TTK</td><td>Histidine (His)</td><td>CAK</td>

<td>Leucine (Leu)</td><td></td><td>xty</td><td>Glutamine (Gin)</td><td>CAJ</td>

<td>Isoleucine (Ile)</td><td></td><td>ATM</td><td>Asparagine (Asn)</td><td>AAK</td>

<td>Methionine (Met)</td><td></td><td>ATG</td><td>Lysine (Lys)</td><td>AAJ</td>

<td>Between (Vai)</td><td></td><td>GTL</td><td>Aspartic acid (Asp)</td><td>GAK</td>

<td>Serine (Ser)</td><td></td><td>QRS</td><td>Glutamic acid (Glu)</td><td>CAJ</td>

<td>Proline (Pro)</td><td></td><td>CCL</td><td>Cysteine ​​(Cys)</td><td>TGK</td>

<td>Threonine (Thr)</td><td></td><td>ACL</td><td>Tryptophan (Try)</td><td>TGG</td>

<td>Alanine (Lower)</td><td></td><td>GCL</td><td>Arginine (Arg)</td><td>WGZ</td>

<td>Tyrosine (Tyr)</td><td></td><td>TAK</td><td>Glycine (Gly)</td><td>GGL</td>

Terminal signal TAJ

Terminal signal TGA

Key: Each three-letter triplet represents a DNA trinucleotide with a 5 'end on the left and a 3' end on the right. The letters represent the purine and pyrimidine bases from which the nucleotide sequence is formed.

A = adenine G = guanine C = cytosine T = thyrine

X = T t-ai C if Y is A or G

X = G 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 if 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 CL = A. T, C or GM = A, C or T

Transcription is the first step in the biological process by which nucleotide sequence information is converted to an amino acid sequence. At this point, the RNA is first copied to the DNA segment whose sequence defines the protein to be produced. RNA is a polynucleotide similar to DNA except that deoxyribose has been replaced by ribose and uracil is used in place of thymine. The bases of RNA can settle in similar base pairs as the bases of DNA. Thus, RNA transcription of a DNA nucleotide sequence is complementary to the sequence to be copied. Such RNA is called messenger RNA (mRNA) because it acts as a mediator between the cellular genetic system and the protein-synthesizing system.

Within a cell, mRNA is used as a template in the complex process involving numerous enzymes and cellular systems that results in the generation of a particular amino acid sequence. This process is called mRNA translation.

Often, there are also additional steps in which the amino acid sequence synthesized in the translation process is converted to a functional protein. Insulin is an example of this.

The immediate precursor of insulin is a single polypeptide, called proinsulin, which contains two insulin chains, A and B, joined by peptide C, cf. Steiner, DF, Cunningham, D., Spigelman, L. and Aten,

B. Science 157, 697 (1967). According to recent information, the original translation product of insulin mRNA is not proinsulin but preproinsulin containing 20 additional amino acids at the amino terminus of proinsulin, cf. Cahn., SJ,

Keim, P. and Steiner DP, Proc. Natl. Acad. Sei. USA, 73, 1964 (1976) and Lomedico, PT and Saunders, GF, Nucl.

Acids. Res. 3, 381 (1976). The structure of preproinsulin can be represented as follows: NH_O(pre-peptides) -chain B- (chain C) -chain A-C00H.

Many proteins of medical or research importance are located in or produced by cells of higher organisms, such as vertebrates. These include e.g. insulin hormone, other peptide hormones such as growth hormone, blood pressure regulating hormones and many hormones of industrial, medical or research importance. These proteins are often difficult to extract from the body in useful amounts, and this problem is quite severe for proteins of human origin. Thus, there is a need for methods by which such proteins can be produced in sufficient amounts in cells outside the organism. In certain cases, it is possible to provide suitable cell lines that can be maintained by tissue culture techniques. However, tissue culture is slow, the medium is expensive, the conditions are precisely controlled, and the yield is low. In addition, it is often difficult to maintain a cell line constant while maintaining the desired specific properties.

In contrast, microorganisms, such as bacteria, are relatively easy to grow in chemically defined media. Fermentation technology is advanced. It is possible to grow organisms quickly and in high yields. In addition, some microorganisms have been thoroughly studied and known for their genes and properties.

Thus, it is highly desirable to be able to transfer the genetic code of a medically significant protein from an organism that normally renders the protein to a suitable microorganism. In this way, the microorganism can produce the protein under controlled growth conditions and the desired amount of protein can be obtained. Manufacturing costs may also be substantially reduced if the protein is prepared by such a method. In addition, the ability to isolate and transfer the genetic sequence defining the production of a particular protein to a microorganism whose genetic background ·, is well known, provides a valuable tool for knowing how the synthesis of this protein is regulated and how the protein is modified after synthesis..Genetic sequences can also be modified to produce proteins with altered therapeutic or functional properties.

The present invention includes a method for achieving the objects described above. This is a multi-step process involving enzyme-catalyzed reactions^-. The nature of these enzyme reactions will be explained on the basis of facts known to date.

Reverse transcriptase (DNA polymerase) catalyzes the synthesis of DNA complementary to the RNA template strand in the presence of the RNA template, DNA template, and four deoxynucleoside triphosphates, dATP, dGTP, dCTP, and dTTP. The reaction is initiated by non-covalent binding of the DNA template to the 3 'end of the mRNA and is followed by the gradual attachment of the required deoxynucleotides to the 3' end of the growing chain as base pairs defined by the mRNA nucleotide sequence. The resulting molecule can be considered as a hairpin structure containing the original RNA linked by a single strand of DNA to a complementary strand of DNA. Reverse transcriptase is also capable of catalyzing a similar reaction using a single-stranded DNA template, the resulting product being a double-stranded DNA hairpin with single-stranded DNA joining the strands at one end, cf. Aviv, H. and Leder, P., Proc. Natl. Acad. Sei. USA 69, 1408 (1972) and Efstratiadis, A., Kafatos, FC, Maxam, AF and Maniatis, T., Cell 7, 279 (1976).

Restriction endonucleases are enzymes capable of hydrolyzing phosphodiester bonds in double-stranded DNA to form a breakpoint in the DNA strand. If the DNA is in the form of a closed loop, the structure of the loop becomes linear. The main property of this type of enzyme is that its hydrolytic effect is limited to a site with a specific nucleotide sequence. This sequence is termed the restriction endonuclease recognition site. Restriction endonucleases have been isolated from a variety of sources and characterized by the nucleotide sequence of their recognition sites. Some restriction endonucleases hydrolyze phosphodiester bonds at the same site in each strand to form a blunt end. Some catalyze the hydrolysis of bonds separated by a few nucleotides, creating a free single-stranded region at each end of the molecule. Such single-stranded ends are self-complementary and thus cohesive and can be used to anneal hydrolyzed DNA. Because a particular enzyme can be predicted to cleave DNA molecules with the same recognition site, the same cohesive ends are generated, and thus it is possible to combine restriction endonuclease-treated heterologous DNA sequences with other similarly treated sequences, cf.

Roberts, RJ Crit. Rev. Biochem. 4, 123 (1976). However, restriction sites are quite rare, but the utility of restriction endonucleases has been enhanced by synthesizing double-stranded digonucleotides with a recognition site sequence. Thus, almost any DNA segment can be ligated to any other segment by attaching the required restriction oligonucleotide to the ends of the molecule, exposing the product to the required restriction endonuclease to create the necessary cohesive ends, see Heyneker, HL, Shine, J., Goodman , HM, Boyer, HW, Rosenberg, J., Dickerson, RE, Narang, SA, Itakura,

K., Lin, S. and Riggs, AD, Nature 263, 748 (1976) and Scheller, RH, Dickerson, RE, Boyer, HW, Riggs,

AD and Itakura, K., Science 196, 177 (1977).

S1 endonuclease is a general enzyme capable of hydrolyzing phosphodiester bonds or single-stranded junctions in single-stranded DNA, cf. Vogt, VM, 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 5'-phosphate and 3'-hydroxyl, respectively, and which may consist of two DNA fragments held together by cohesive ends. The normal mode of action of an enzyme is considered to be that it combines several daughter DNA fragments generated in parallel with another strand. However, DNA ligase can, under suitable conditions, catalyze the fusion of blunt ends in which two blunt-ended molecules covalently join, cf. Sgaramella, V., Van de Sande, JH and Khorana, HG, Proc. Natl. Acad. Sei. USA 67, 1468 (1970).

Alkaline phosphatase is a general enzyme capable of hydrolyzing phosphate esters, including the 5 'terminal phosphates of DNA.

In a sub-step of the method of this invention, a particular DNA fragment is inserted into a DNA vector, such as a plasmid. Plasmid is the name used for any independently, replicating unit of DNA that is present in a microbial cell and does not belong to the host cell's own genome. The plasmid is not genetically bound to the chromosome of the host cell. Plasmid DNA exists as circular double-stranded molecules, usually with a molecular weight of a few million, some even more than 10, and usually represent only a small percentage of the total DNA of the cell. Plasmid DNA can usually be separated from host cell DNA due to its large size difference. Plasmids can replicate regardless of the rate of division of the host cell, and sometimes their rate of division can be regulated by altering growth conditions. Although the plasmid exists as a closed ring, a DNA segment can be artificially inserted to form a recombinant plasmid with a higher molecular weight whose replication capacity and gene expression capacity are not substantially altered. Thus, the plasmid serves as a useful vector to transfer a DNA segment to a new host cell. Plasmids suitable for recombinant DNA technology are particularly those that contain genes suitable for selective purposes, such as genes that confer drug resistance.

To illustrate the practice of this invention, the isolation and transfer of the rat insulin gene will be described in detail. Insulin was chosen as the target because of its central role in clinical medicine and basic research. Those skilled in the art will be able to isolate the insulin gene from an organism, including a human, by the method described.

Insulin was first isolated in 1922. Today, the use of this hormone in the treatment of diabetes is well known. Although bovine and porcine pancreas from slaughterhouses are a source of insulin, there will be a shortage of this hormone as the number of diabetics in the world increases. In addition, some diabetics develop a harmful allergy to bovine and porcine insulin. Therefore, it would be highly desirable to be able to produce sufficient human insulin to meet a global need. If human insulin could be produced by bacteria, this goal would be achievable. Until the present invention, however, the goal has been hampered by the fact that there has been no method by which the insulin gene can be transferred to bacteria.

The method according to the invention is characterized in that

a) the mRNA is isolated from the islet cells of the pancreatic tissue by homogenization in the presence of a ribonuclease inhibitor, for example a mixture of 4-m guanidium thiocyanate and 0.05 to 1.0-m / 3-mercaptoethanol at pH 5.0 to 8, 0, so that no ribo nuclease degradation of the mRNA occurs,

b) preparing a cDNA having a nucleotide sequence encoding insulin from the obtained mRNA by means of a reverse transcriptase enzyme in a manner known per se, and said cDNA being double-stranded,

c) ligating the restriction endonuclease recognition site sequences to the ends of the resulting double-stranded cDNA in a manner known per se, wherein the restriction endonuclease has the same enzyme as in d) below, after which the cDNA is cleaved by said restriction endonuclease to form cohesive ends,

d) opening the bacterial plasmid vector with a restriction endonuclease, for example Hind III, Hsu I or Eco RI, in a manner known per se, e.g. by incubating at pH 7.6 at 37 ° C for 2 hours to obtain the opened plasmid vector, with cohesive ends,

e) hydrolysing the 5'-phosphate end groups of the plasmid vector obtained in d) by treatment with, for example, alkaline phosphatase at pE 8.0 at 65 ° C for 30 minutes, whereby the cohesive ends mentioned in d) cannot join together,

f) coupling the plasmid vector treated according to e) and the cDNA obtained in c) in a manner known per se by incubation in the presence of DNA ligase and ATP, e.g. at pH 7.6 at 14 ° C for one hour, using a molar excess of the plasmid vector, to obtain a recombinant DNA molecule comprising a nucleotide sequence encoding insulin.

A practical aspe ct of the method of the present invention will be elucidated by describing the isolation, transfer into bacteria, and replication of the nucleotide sequence of rat insulin. Similarly, the nucleotide sequence of rat growth hormone has been isolated, transferred, and replicated in bacteria. The method is also suitable for transferring a nucleotide sequence isolated from a human, such as human insulin and growth hormone, as well as other polypeptide hormones.

This invention includes a method by which a DNA molecule having a particular nucleotide sequence can be isolated and transferred to a microorganism, and the original nucleotide sequence of the DNA found after replication in the organism.

The various steps of the method contained in this invention can be divided into four groups.

1. Isolation of the desired cell from a higher organism

There are two possible sources for the genetic code of a particular protein, namely the DNA of the source organism or the RNA transcription of the DNA. According to the current safety requirements of the US National Institutes of Health, human genes, whatever they may be, can be inserted into recombinant DNA and then into bacteria only when the genes have been thoroughly purified or when there are particularly high-risk workplaces available. spaces (P4), see Federal Register, Vol. 41, no. 131, 1967-07-07, pp. 27902 - 27943. Preferably, the starting point is to isolate a specific mRNA that contains the code of the desired protein. This approach also has the advantage that mRNA extracted from the cell can be purified more easily than DNA extracted from the cell. In particular, it is possible to use the fact that highly specialized organisms, such as vertebrates, have certain cells in certain places that are responsible for producing some of the protein in question. Alternatively, such a cell may be present at some stage in the development of the organism. Most of the mRNA isolated from the cells of such a cell contains the desired nucleotide sequence. Thus, the choice of cell to be isolated and method of isolation can take into account the advantages offered by the initial degree of purity of the mRNA to be isolated.

In most tissues, glands, and organs, cells are joined by a fibrous connective tissue that is composed primarily of collagen, but may also contain other structural proteins, polysaccharides, and mineral stores, depending on the tissue. Isolation of cells from a particular tissue requires methods by which the cells can be detached from the connective tissue. Isolation and purification of a particular specialized cell type thus involves two main steps, which are to separate the cells from the connective tissue and to separate the desired cell type from other types of tissue cells. As an example, a method for isolating pancreatic islets of Langerhans suitable for isolating insulin mRNA is described.

Insulin-producing cells can also be derived from other sources, such as the pancreas of a fetal calf or cultured islet tumor cells. Isolation of pure islet cells is then much simpler, especially if pure cell cultures are used. The method of isolating islet cells described above would then not be needed, but is nevertheless a preferred method because of its general utility.

It can often be seen that the proportion of the desired mRNA can be increased if the cell's ability to respond to external stimuli is exploited. For example, hormone treatment may cause an increase in the production of the desired mRNA. Other methods include cultivation at a certain temperature and / or in a certain nutrient or other chemical substance. When isolating rat growth hormone mRNA, treatment of cultured rat pituitary cells with thyroid hormone and glucocorticoids synergistically significantly increased the amount of growth hormone mRNA.

2. mRNA extraction

An important feature of the present invention is that the Rbiase activity is virtually completely removed from the cell extract. The mRNA to be extracted is a single-stranded polynucleotide with no complementary strand. Thus, hydrolytic cleavage of any phosphodiester bond in the sequence would render the entire molecule incapable of transferring the intact genetic sequence to the microorganism. As mentioned above, the RN donkey is widespread and is very active and exceptionally stable.

It is on the skin, it withstands the usual methods of washing glassware and sometimes it contaminates organic chemicals. The difficulty in handling pancreatic cell extracts is very great, as the pancreas produces digestive enzymes and is thus highly RNase-rich. However, RNase eruption applies to all tissues and the method of destroying RNase activity described herein can be applied to all cells. The exceptional efficiency of the method is demonstrated in the isolation of unchanged mRNA from pancreatic, islet cells.

The present invention uses a combination of a chaotropic anion, a chaotropic cation, and a disulfide bond cleavage reagent during cell disruption and during all operations required to obtain virtually protein-free RNA. The efficiency of the interaction of the above reagents has been demonstrated in practice by isolating virtually intact mRNA in good yield from islets of Langerhans isolated from rat pancreas.

The choice of suitable chaotropic ions depends on their water solubility and availability. Suitable chaotropic anions include e.g. guanidinium, carbamoylguanidinium, guanylguanidinium, lithium, etc. Suitable chaotropic anions include e.g. iodide, perchlorate, thiocyanate, diiodic salicylate, etc. The relative effectiveness of the salts formed by such anions and cations is determined by their solubility. For example, lithium diiodic salicylate is a more potent denaturant than guanidinium thiocyanate, but has a solubility of only about 0.1 M and is also relatively expensive. Guanidinium thiocyanate is the preferred cationic anion combination because it is readily available and has good solubility in aqueous solutions, up to about 5 M.

Thiol compounds, such as β-mercaptoethanol, are known to disrupt the intramolecular disulfide bonds of proteins by the thiol disulfide exchange reaction. In addition to β-mercaptoethanol, several suitable thiol compounds are known, such as dithiothreitol, cysteine, propanol dimercaptan, etc. Water solubility is a necessary requirement, as a large excess of thiol compound must be present compared to intramolecular disulphides in order for the exchange reaction to be practically complete. γ-mercaptoethanol must be given priority because it is readily available at a reasonable price.

The efficacy of a particular chaotropic salt is directly proportional to its concentration when it is desired to inhibit RNase by extracting RN / v from cells or tissues. The preferred concentration is therefore the highest concentration that can be practiced. The success of the present invention in keeping the mRNA intact during extraction is thought to be due to the rate at which the RNase is denatured and the degree of denaturation. This apparently explains the superiority of guanidinium thiocyanate over hydrochloride, although the hydrochloride is only slightly weaker as a denaturant. The potency of a denaturant is therefore defined as the threshold concentration required for complete denaturation of the protein. On the other hand, the rate of denaturation of proteins often depends on the ratio of the denaturant concentration to a threshold value of 5 to 10 raised to the power, cf. Tanford, CA, Adv. Prot. Chem. 23, 121 (1968). Qualitatively, this ratio means that only a slightly more efficient denaturant than guanidinium hydrochloride can denature the protein many times faster at the same concentration.

The use of a disulfide cleavage reagent in combination with a denaturant enables and enhances the latter effect because the RNase molecule becomes fully opened. The thiol compound is thought to aid in the progress of the denaturation process because it prevents the rapid denaturation that can occur if intramolecular disulfide bonds are left intact. In addition, the RNase impurity contained in the mRNA preparation remains virtually inactive even in the absence of denaturant and thiol. Disulfide cleavage reagents with thiol groups are somewhat effective at any concentration, but it is preferable to use a large excess of thiol groups over intramolecular disulfide bonds, as this will drive the exchange reaction toward the cleavage of intramolecular disulfide bonds. On the other hand, since many thiol compounds are malodorous and it is uncomfortable to work with high concentrations, there is an upper concentration limit for practical reasons. When β-mercaptoethanol is used to separate intact RNA from rat pancreas, concentrations in the range of 0.05-1.0 M have been found to be effective, and the optimal concentration is considered to be 0.2 M.

The pH of the medium can be anywhere from 5.0 to 8.0 when the mRNA is extracted from the cells.

After cell disruption, RNA is separated from cellular proteins and DNA. Several methods have been developed for this purpose, all of which are suitable and known to those skilled in the art. A common method previously used is ethanol precipitation, with which RNA is selectively precipitated. In the process of this invention, it is more preferable to omit the precipitation step and deposit the homogenate directly in a 5.7 M cesium chloride solution in a centrifuge tube and then perform centrifugation as described in Glisin, V., Crkvenjakov, R. and Byus, C., Biochemistry 13, 2533 (1974 ). This method is advantageous because the environment harmful to RNase can be preserved at all times and RNA free of DNA and protein is obtained in good yield.

By the method described above, the entire RNA of the cell homogenate is purified. However, only a portion of this RNA is the desired mRNA. Further purification takes advantage of the fact that in cells of higher organisms, mRNA is processed after transcription by the addition of polyadenyl acid. Such mRNA containing poly-A sequences can be selectively separated on a chromatography column packed with cellulose to which oligothymidylate has been added, cf. Avis, H. and Leder, P., supra. The method described above is suitable when virtually pure, undamaged and translatable mRNA from RNase-rich sources is required.

In certain cases, such as when using tissue culture cells as a source of mRNA, the RNase impurity may be so low that the RNase inhibition described above is not required. In this case, the previously known methods for removing RNase activity are sufficient.

3. cDNA generation

In this context, reference is made to Figure 1, which is a schematic representation of the remaining steps of the method. The first step now is to generate a DNA sequence complementary to the purified mRHA. Reverse transcriptase is selected as the enzyme for this reaction, although in principle any enzyme capable of forming a complementary strand of DNA could be used using mRNA as a template. The reaction can be performed under conditions known in the art by using mRNA as a template and a mixture of four deoxynucleoside triphosphates as a precursor of the DNA strand. It is preferred that one of the phases of the deoxynucleoside triphosphine 3 is labeled at the ot position with a P atom, as it can be used to monitor the reaction, it serves as a marker in separation methods such as chromatography and electrophoresis, and it can be used to draw quantitative conclusions. Efstratiadis, A., et al., Supra.

As shown, the reverse transcriptase reaction result is a double-stranded hairpin structure in which the RNA strand and the DNA strand are joined together by a non-covalent bond.

The product of the reverse transcriptase reaction is removed from the reaction mixture by known methods. It has been found useful to use a combination of phenol extraction, chromatography (Sephadex G-100, Pharmacia Inc., Uppsala, Sweden) and ethanol extraction.

Once the cDNA has been synthesized enzymatically, the RNA template can be removed. Several methods are known for selectively degrading RNA in the presence of DNA. Base hydrolysis is a preferred method because it is highly selective and can be easily adjusted by pH.

After emashydrolysis and neutralization, the P-labeled cDNA can be concentrated by ethanol precipitation if desired.

Such double-stranded hairpin cDNA is synthesized by a suitable enzyme. such as ENA nolymerase or reverse transcriptase. The reaction conditions are as previously described. . . . .

«Tr P-labeled nucleoside triphosphate. Encapsulated transcriptase is obtained from several sources. Avian myeloblastosis virus is a preferred source. The virus is available from Dr. D. J. Beard 'ilt. Life Sciences Incorporated, St. Petersburg, Florida ··¹, which makes the virus under an agreement with the National Institutes of Health.

Once the cDNA hairpin is formed, it may be advantageous to purify it from the reaction mixture. As previously mentioned, it has been found advantageous to use phenol extraction, chromatography (Sephadex G-1CO) and ethanol precipitation when it is desired to obtain DNA purified from protein impurities.

the hairpin structure can be transformed into a common double-stranded DNA structure by removing a single strand connecting the complementary strands. There are several enzymes capable of specifically hydrolyzing single-stranded portions of DNA. A well-suited enzyme for this purpose is the S1 nuclease isolated from Aspergillus oryzae (manufactured by Miles Research Products, Elkhart, Indiana). Treatment of the DNA hairpin structure with S1 nuclease yields molecules with base pairs in good yield. Extraction, chromatography and ethanol precipitation are then performed as described above. Efstratiadis et al. have described in the above publication the synthesis of double-stranded cDNA transcripts of mRNA by reverse transcriptase and S1 nuclease.

The proportion of blunt-ended cDNAs may optionally be increased if treated with E. coli DNA polymerase I in the presence of four deoxynucleoside triphosphates. The interaction between the exonuclease activity and the polymerase activity of the enzyme acts to remove the protruding 3 'end and fill the protruding 5' end. This ensures that · as many cDNAs as possible are involved in the next step ligation reactions.

In the next step of the method of the invention, the ends of the cDNA product are processed so as to obtain a restriction endonuclease recognition site sequence at each end. Practical reasons determine the choice of DNA fragment to be attached to the ends. The sequence to be inserted at the ends is selected based on the restriction endonuclease, which in turn is selected based on the DNA vector to which the cDNA is inserted. The plasmid of choice should have at least one site to which the restriction endonuclease cleavage effect may be directed. For example, plasmid pMB9 contains a single recognition site for the enzyme Hind III. Hind III is isolated from Haemophilus influenzae and purified by the following method: Smith, HO and Wilcox,

KW, J.Mol.Biol. 51, 379 (1970). The enzyme Hae III from Haemophilus aegyptious is purified by the following method ·. Middleton, JH, Edgell, MH and Hutchison III, CA, J. Virol. 10, 42 (1972).

The enzyme from Haemophilus suis, Hsu I, catalyzes the same site-specific hydrolysis at the same recognition site as Hind III. Thus, these two enzymes can be considered functionally alternative.

For insertion into the ends of a double cDNA, it is preferred to use a chemically synthesized double-stranded decanucleotide containing a Kind III recognition site. The sequence of the double-stranded decanucleotide is shown in Figure 1. Heyneker, HL, et al. and Scheller, RH, et al. above. Several such recognition site sequences are available to those skilled in the art, and it is therefore possible to select the ends of the double DNA to suit the restriction endonuclease required in each case.

The insertion of restriction site sequences into the ends of the cDNA can be accomplished by a method termed blunt end insertion and is catalyzed by a DNA ligase purified by the following method: Panet, A., et al., Biochemistry 12, 5045 (1973). The aforementioned Sgaramella, V., et al. have described the blunt-end coupling reaction. Coupling at the blunt end, where the reaction takes place with a blunt-ended cDNA and a large molar excess of a double-stranded decanucleotide containing a Hind III endonuclease recognition site, yields a cDNA with a Hind III restriction site sequence at each end. When the reaction product is treated with Hind III endonuclease, cleavage occurs at the restriction site and single-stranded self-complementing 5 'ends are formed, as shown in Figure 1.

4. Construction of a recombinant DNA transfer vector

Suitable transfer vectors currently approved for use are ram. several bacteriophage lambda derivatives (see, e.g., Blattner, FR, Williams, BG, Blechl, Α.Ξ., Denniston-Thompson, K., Faber, HE, Furlong, LA,

Grunwald, DJ, Kiefer, DO, Moore, DD, Schuram, JW,

Sheldon, EL and Smithies, 0, Science 196, 161 (1977)) and col E1 plasmid derivatives (see e.g. Rodriguez, RL, Bolivar, S., Goodman, HM, Boyer, HW and Betlach, MN, ICN- The UCLA Symposium on the Ecological Mechanism in Control of Gene Expression, DP Nierlich, WJ Rutter, CF Fox, Eds. (Academic Press, NY, 1976) p.

471-477). Plasmids derived from Col E1 are characterized by a relatively small size, having a molecular weight of the order of a few million, and the fact that under normal conditions the number of copies of plasmid DNA per host cell is 20-40, but can be raised to a thousand or more when host cells treated with chloramphenicol. The ability of a host cell to increase the number of genes it contains, under certain conditions, under the control of a researcher, allows the host cell to produce primarily proteins encoded by plasmid genes. Thus, such col E1 derivatives are preferred transfer vectors in the method of this invention. Suitable col E1 derivatives include e.g. plasmids pMB-9, the gene of which confers resistance to tetracycline, and pBR-313, pBR-315, pBR-316, p3R-317 and pBR-322, which contain the tetracycline resistance gene and the ampicillin resistance gene. The presence of the resistance-causing gene provides a suitable means of selecting cells that are infected with the plasmid, as such colonies grow in the presence of the drug, but in the absence of the plasmid, the cells do not grow. In the examples contained in this description, a plasmid derived from col E1 containing the em resistance gene and one Hind III recognition site was used.

Plasmid pBR-322 has been highly characterized.

Bolivar, F. et al. (Gene 2 (1977) 95) has described the synthesis and characterization of this plasmid. Plasmidin g

pBR322 has a molecular weight of 2.7 x 10 daltons (Bolivar F.,

Gene 4 (1978) 121) and contains the genes causing ampicillin resistance R sin (Aβ) and tetracycline resistance (Te). The aforementioned Aβ gene has one endonuclease Pst 1 recognition site. The Te gene has one recognition site for each of the following endonucleases:

Price III, Sal I and Bam HI. In addition, pBR322 contains one Eco R1 recognition site, two Hind II recognition sites, five Eco RII recognition sites, three Bgl I recognition sites, 12 Alu I recognition sites, 12 Hae II recognition sites, and 17 Hae III recognition sites. identification point. The recognition sites are labeled on the circular plasmid pBR322 on page 103 R

Bolivar et al. in the publication. The Aβ gene is lost upon cleavage of the plasmid from the Pst I recognition site and insertion of p

foreign DNA into it. Similarly, Te is lost upon cleavage of pBR 322 by Hind III, Val I, or Bam I endonucleases and insertion of foreign DNA into their recognition site.

Recombination of DNA into the Pst I recognition site produces recombination molecules that can be detected in strains that

S · · are both ampicillin-sensitive (Aβ) and tetracycline-resistant (Te), and by inserting DNA into the Sai I or Bam HI recognition site of Hind III, recombination molecules are formed that can be detected from strains that are both ampicillin-resistant and tetracycline-sensitive. A transfer vector containing foreign DNA fused to one of the four recognition sites mentioned above can be characterized by the drug resistance properties of the transfer vector, i.e., whether it is ampicillin-sensitive and tetracycline-resistant or ampicillin-resistant and tetracycline-sensitive. The transfer vector can be further characterized by removing the foreign DNA attached to it, determining the molecular weights of the two products formed, and comparing these to the molecular weights of the starting materials. In addition, the characterization can be supplemented by labeling the recognition sites of different endonucleases in the transfer vector. The sequence of the transferred DNA molecule can also be determined. The entire nucleotide sequence of said plasmid pBR322 is shown in J. Sutcliffe's dissertation Nucleotide Sequence of pBR322, Harvard University, Cambridge, Massachusetts, USA.

Similarly, plasmid pBR313 has been highly characterized. The synthesis and characterization of this plasmid has been described by Bolivar, F. et al. (Gene 2 (1977) 75).

β

Plasmid pBR313 has a molecular weight of 5.8 x 10 daltons and contains genes that confer ampicillin resistance (Aβ) and tetracycline resistance (Te). Plasmid p pBR313 Te has one recognition site for each of the following nucleases: Hind III, Sal I, and Bam HI. The recognition sites of plasmid pBR313 for the various endonucleases have been completely determined, and the resulting recognition site map is shown on page 84 by Bolivar et al. in the publication. When ligating foreign DNA into Hind III.

At the recognition site of Sai 1 or Bam HI, tetracycline resistance disappears. Thus, recombinant DNA molecules are found

4 64 0 strains that are both ampicillin-resistant and tetracycline-sensitive. The transfer vector can be characterized on the basis of drug resistance properties, the transferred DNA sequence, the restriction enzyme recognition site map, and the molecular weights mentioned above.

The third plasmid that has been highly characterized is pMB 9. This plasmid was prepared according to Rodriguez, RL et al. (above) and Bolivar, F.

et al. (Gene 2 (1977) 75). Plasmid pMB 9 θ has a molecular weight of 3.5 x 10 daltons and contains the gene that causes tetracycline resistance (Te). This plasmid contains one recognition site for each of the following endonuclei^a§^ei^stai Eco ri, Price III, Sal I and

Bam HI. Of these, the recognition sites of the latter three are shared in the Te gene. When foreign DNA is ligated into the Hind III, Sai I, or Bam HI recognition site, tetracycline resistance disappears. A transfer vector containing foreign DNA at some of these sites can be characterized by this property. This transfer vector can be further characterized by sequencing the transferred DNA molecule, comparing molecular weights, and by recognition site analysis as described above.

Plasmid pSCIOI has been highly characterized. Cohen,

SH et al. (Proc. Nat. Acad. Sci. USA 70 (1973) 1293) have described the synthesis and preliminary characterization of this plasmid. Characterization has been supplemented by Cohen, SN et al., Proc.Nat.Acad. Sci. USA 70 (1973) 3240 Boyer, HW et al. (Recombinant Molecules), Beers, RF and Basset, EG ed., Raven Press, New York, s 13 (1977) and Cohen, SN et al (Recombinant Molecules, p. 91).

β

Plasmid pSCIOI has a molecular weight of 5.8 x 10 daltons,

R and contains the gene that causes tetracycline resistance (Te). The Te gene contains one recognition site for each of the following endonucleases: Hind III, Sal I, and Bam HI. In addition, plasmid pSCIOI contains one Eco RI recognition site, one Hpa I recognition site, one Sma I recognition site, and four Hinc II recognition sites. Tetracycline resistance is lost by cleavage of pSCIOI with Hind III, Sal I or Bam HI endonucleases and insertion of foreign DNA into their recognition site. In Appendix 22 to the DNA Hind III, Sa I or Bam HI recognition site, recombination molecules are found in cultures that are tetracycline sensitive. A transfer vector containing foreign DNA ligated to one of the three sites mentioned above can be characterized based on the resistance properties of the transfer vector. The transfer vector can be further characterized by (a) deleting the transferred foreign DNA sequence, determining and comparing molecular weights, (b) constructing a recognition site map, and (c) determining the sequence of the transferred DNA molecule.

Several vectors can be used to transform Bacillus subtilis. These vectors are derived from the microorganism Staphylococcus aureus (see Fordanescu, S.,

J. Bacteriol. 124 (1974) 597 and Ehfllch, SD, Pröc.

Nat. Acad. Sei. USA, 74 (1977) 1680). These plasmids have specific resistance and specific restriction endunuclease recognition sites. For example, plasmid pC194 confers resistance to chloramphenicol, (Cm), contains a single Hind III site, and has a molecular weight θ

is 1.8 χ 10 daltons. The foreign DNA thus binds to the Hind III site of plasmid pC194. The transfer vector containing the foreign DNA can be characterized by its resistance baseline (Cm). The transfer vector can also be characterized by removing the ligated DNA by determining the molecular weight of the two products, compared to the molecular weight of the starting materials. The sequence of the ligated DNA can also be determined.

Recombinant plasmids are constructed by mixing restriction endonuclease-treated plasmid DNA and cDNA with end groups treated accordingly. Plasmid DNA is used in a large molar excess over the cDNA to minimize combinations of cDNA segments with each other.

It is preferred to use a method that minimizes the number of colonies from which the recombinant plasmid is sought.

The method is performed by treating restriction endonuclease-digested plasmid DNA with alkaline phosphatase (manufactured by Worthington Biochemical Corporation, Freehold,

New Jersey). Alkaline phosphatase removes phosphates at the 5 'end from the ends of the plasmid generated by the endonuclease and prevents self-ligation of the plasmid DNA. Thus, ring formation and thus transformation depends on the incorporation of a DNA fragment containing 5'-phosphorylated ends. The method described reduces the relative incidence of non-recombinant transformation to less than 1-10.

The present invention is based on the fact that the reaction catalyzed by DNA ligase takes place between the 5 'phosphate end group of DNA and the 3¹between the hydroxyl end group. In the absence of 5'-phosphate, no coupling reaction occurs. When double-stranded DNAs need to be combined, there are three situations, as shown in Table 1.

apaus

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Ligase product

3'

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II

III

table 1

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O? O₃B₇^{+ 2 3}ho3 '

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5’

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5'

3'

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J-2H₂o + h₂°

In Table 1, double-stranded DNA is shown schematically as solid parallel lines and their respective 5 'and 3' end groups are labeled as hydroxyls (OH) or phosphates (OPOjH?). In case I, both reactive molecules have 5'-phosphate at their ends, and thus both strands covalently join each other. In case II, only one of the ends to be joined contains 5'-phosphate and thus only one of the strands to be joined is covalently attached to the other and a discontinuity point remains in the other strand. The covalently unjoined strand remains associated with the joined strand by hydrogen bridges present between the complementary base pairs of the opposite strands. In the case of ITI, there is no 5'-phosphate at either reactive end and no fusion reaction occurs.

Thus, the 5 'phosphate group must be removed from the end whose attachment to the other is to be prevented.

Any method of removing the 5 'phosphate group that does not otherwise damage the DNA structure is suitable for use.

Preferably, alkaline phosphatase catalyzed hydrolysis is used.

The method described above is also useful in the case where it is desired to cleave a linear DNA molecule into two subfragments, usually using a restriction endonuclease, and then reconstruct the original sequence. The subfragments can be purified separately and the desired sequence can be reconstructed by combining the subfragments. For this purpose, a DNA ligase can be used which catalyzes the joining of the ends of the DNA fragments to each other, cf. Sgaramella, V., Van de Sande, JH and Khorana, HG, Proc. Natl. Acad. Sei.

USA 67, 1468 (1970). If the sequences to be joined are not blunt-ended, ligase from E. coli can be used, cf. Modrich, P. and Lehman, IR, J. Biol. Chem 245, 3626 (1970).

Reconstruction of the original sequence from subfragments obtained by restriction endonuclease treatment is greatly enhanced if a method can be used which avoids the reconstruction of inappropriate sequences. An inappropriate result can be avoided if a homogeneous length cDNA fragment having the desired sequence is treated with a reagent capable of removing 5 'terminal phosphate groups from the cDNA before the homogeneous cDNA is cleaved by restriction endonuclease. Here, alkaline phosphatase is the preferred enzyme. 5¹terminal phosphates are a structural prerequisite for the binding activity of DNA ligase subfragments. Thus, ends that do not have a 5 'terminal phosphate cannot be covalently joined. DNA subfragments can only be joined to ends that contain a 5 'terminal phosphate formed as a result of the cleavage effect of the restriction endonuclease on the isolated DNA.

The procedure described above prevents the most inappropriate joining reaction, namely that the two fragments are joined in the reverse order, i.e. the back to the front and not the front to the back. Other possible side reactions, such as dimerization or ring formation, cannot be prevented because these occur according to reaction type II, cf. Table 1 above. However, such side reactions are less detrimental because they result in physically identifiable and separable products, whereas reverse recombination does not.

To illustrate the methods described above, the rat insulin cDNA code has been isolated and recombined with the plasmid. The bacterial strain transformed by the DNA molecules was E. coli X-1776. Transformed bacteria were isolated by culturing in tetracycline-containing medium. The DNA of a recombinant plasmid contained in the transformed cells was found to contain an associated DNA fragment of approximately 410 nucleotides in length. Other recombinants were also isolated and analyzed by the same method. The ligated fragments were excised from the plasmid by Hind III or Hsu I endonuclease treatment and their DNA sequence was analyzed by the following method: Maxam, AM and Gilbert, W., Proc. Natl. Acad. Sei.

USA 74, 560 (1977). The nucleotide sequence of the ligated DNA fragments was found to be too long and contained the code for rat whole rat proinsulin I and the prepeptide sequence for thirteen amino acids, for a total of 23 amino acids. The following is the nature of this nucleotide sequence.

The method just described can be used to isolate and purify a gene of a higher organism, including a human gene, and transfer it to a microorganism in which it replicates. The description describes novel recombinant plasmids containing all or part of the isolated gene.

Example 1

This example describes the extraction and isolation of rat insulin mRNA, the synthesis and characterization of DNA complementary thereto. To prepare rat islet cells, the pancreas of anesthetized rat was infused with Hank's saline by infusing the posterior pancreatic duct. Hank's saline solution is a standard solution known to those skilled in the art, which is sold e.g. Grand Island Biological Supply Company,

Grand Island, New York. The pancreas was removed, ground in Hank's solution at 0 ° C, and the trypsin inhibitor of collagenase and soybean was allowed to act. All procedures were performed at 0-4 ° C unless otherwise indicated. The circumstances of the latter measure were very critical. Two ground rat pancreases in Hank's solution were placed in a 30 ml glass tube to a total volume of 8 ml. All glass tubes were pretreated with silicone (Siliclad, Clay-Adams Division, Becton-Dickinson Inc., Parsippany, New Jersey). The incubation mixture contained 12 mg of collagenase, an enzyme prepared from Clostridium histolyticum (method of preparation: Mandi, I., Mackennan, JD and Howes, EL, J. Clin. Invest. 32, 1323 (1943)), type CLS IV, obtained from Worthington Biochemical Corporation, Freehold,

New Jersey, and 1 mg of soybean trypsin inhibitor available from Sigma Chemical Company, St. Louis, Missouri. The tube was shaken at 90 shakes per minute at 37 ° C for 25 minutes. The optimal progression of collagenase activity had to be monitored continuously.

If the incubation was too short, the detachment of the islet cells remained incomplete, and if the incubation time was too long, the islet cells began to dissolve. After incubation, the tube was centrifuged for 1 minute at 200 rpm. The mother liquor was decanted and the particles were washed with Hank's solution, and the centrifugation and washing were repeated a total of five times. After final centrifugation, the particles were suspended in any Ficoll solution (Pharmacia Chemical Company, Uppsala, Sweden) with a density of 1.085. Then 8 ml of Ficoll solution with a density of 1.080 and 5 ml of Ficoll solution with a density of 1.060 were added.

2% and the tube was serially centrifuged in a swinging cup rotor first for 5 minutes at 500 rpm and then for 5 minutes at 2000 rpm. As a result of this procedure, the vesicle cells remained at the bottom of the tube and the islet cells rose to the gradient and formed a front between the top two layers. The islet cell layer contained neurons, lymph nodes, and connective tissue as impurities. Large impurity particles were removed from the material in the bed. The remainder of the material was placed in an adventure microscope, and visible impurities were removed by hand with a micropipette. The cell preparation was then mixed with Hank's solution and centrifuged. The mother liquor was decanted and the cellular material was stored in liquid nitrogen.

200 rat islet cells were homogenized at 4 ° C in 4M guanidinium thiocyanate (Tridom,

Fluka AG Chemische Fabrik, Buchs, Switzerland) containing 1 My&gt; mercaptoethanol and buffered to pH 5.0. The homogenate was layered with 1.2 ml of 5.7 M CsCl containing 100 mM EDTA and centrifuged at 15 ° C for 18 hours at 37,000 rpm in a Beckman ultracentrifuge rotor SW 50.1 (Beckman Instrument Company Fullerton, California). RNA migrated to the bottom of the tube.

Polyadenylated RNA was isolated from the whole RNA preparation by chromatography on oligo (dT) cellulose by the method of Aviv, H. and Leder, P., cf. above.

All polyadenylated RNA from rat Langerhans islets was transcribed into cDNAs using avian myeloblastosis virus reverse transcriptase obtained from Dr. DJ Beard (Life Science Inc., St. Petersburg, Florida). The reaction was performed in a mixture of 50 mM Tris-HCl, pH 8.3, 9 mM MgCl 2, 30 mM NaCl, 20 mM β-mercapto · ethanol, 1 mM each of three non-radioactive deoxyribonucleoside triphosphates, 250 μM fourth deoxynucleoside triphosphate labeled with Ot-P with a specific activity of 50-200 Ci / mcl, 20 ug / ml oligo-dT - ^ pg / (Collaborative Research, Waltham, Massachusetts), 100 ug / ml polyadenylated RNA: and 2 00 units / ml reverse transcriptase. The mixture was incubated at 45 ° C for 15 minutes. EDTANa was then added to a concentration of 25 mM, the solution was extracted with a self-contained volume of phenol saturated with water, and then the aqueous phase was chromatographed on a Sephadex G-10O column (diameter 0.3 cm, height 10 cm) using eluent. with 10 mM Tris-HCl, pH 9.0,

100 mM NaCl and 2 mM EDTA. Interstitial volume is likely to elute ·? To this nucleic acid was added ammonium acetate, pH 5.0, to a concentration of 0.25 M, and precipitated with ethanol. The precipitate was collected by centrifugation, dissolved in 50 μl of freshly prepared 0.1 M NaOH and incubated for 20 minutes at 70 ° C so that the RNA was hydrolyzed.

The mixture was neutralized by the addition of 1M sodium acetate, pH 4.5, 3 2 and the P-cDNA product was precipitated with ethanol and dissolved in water. A portion of the single-stranded cDNA was analyzed on a native polyacrylamide gel by the following method: Dingman,

CW and Peacock, AC, Biochemistry 7, 659, (1968).

2

The gels were dried and the&gt; DNA was examined by autoradiography using Kodak No-Screen NS-2T film (Eastman Kodak Corporation, Rochester, New York). Judging by the electrophoresis pattern, the cDNA was heterodisperse. Judging by known standards, it contained at least one cDNA species of about 450 nucleotides.

Example la

Example 1 was repeated, however, using different concentrations of β-mercaptoethanol in the islet cell homogenization step, namely 0.05 M, 0.2 M, 0.6 M and 0.8 M. All of these concentrations gave the same result, i.e. no degradation of mRNA by RNasease. happened.

Example Ib

Examples 1 and 1a were repeated, but using different pH in the islet cell homogenization step, namely pH 6.0, 7.0 and 8.0. The same result was obtained at all these pH values, i.e. no degradation of the mRNA by RNase occurred.

Example 2

This example describes the synthesis and characterization of rat insulin double-stranded cDNA. To synthesize the complementary strand, the single-stranded cDNA obtained in Example 1 was treated with reverse transcriptase.^Reak1 -^ioseos contained 50 mM Tris-HCl, pH 3.3, 9 mM MgCl_?, 10 mM dithiothreitol, 50 mM each of three non-radiolabeled deoxyribonucleoside triphosphates, 1 mM oi-P-labeled nucleoside triphosphate with a specific activity of 1-10 Ci / mmol, BO ug / ml cDNA and 220 units / ml reverse transcriptase. The reaction mixture was incubated for 120 minutes at 45 ° C. The reaction was quenched by the addition of EDTA-Na 2 to a concentration of 25 mM, extracted with phenol, chromatographed on Sephadex G-100 and precipitated with ethanol. A batch of the reaction product having an activity of 500-1000 beats per minute was analyzed by gel electrophoresis as described in Example 1. Comparison with standard samples revealed a heterodisperse band of approximately 450 nucleotides in length. An aliquot of the DNA reaction product of Example 1 and Example 2 was treated separately with restriction endonuclease Hae III and similar gel electrophoresis was performed. Endonuclease cleaved both products and two radioactive bands were detected by gel electrophoresis. The bands generated by cleavage of the double-stranded cDNA represented products of substantially the same length as those generated by cleavage of the single-stranded cDNA.

Example_3

This example describes how the Hind III decanucleotide recognition site is ligated to the double-stranded rat islet cDNA prepared in Example 2 by blunt end ligation. The double-stranded reaction product of Example 2 at a concentration of 2-5 μg / ml was first treated for 30 minutes at 22 ° C and then for 15 minutes at 10 ° C with 30 units of S 1 nuclease at 1200 units / ml (Miles Laboratories , Elkhart, Indiara), in a solution of 0.03 M sodium acetate, 4.5, 0.3 M sodium chloride and 4.5 mM ZnCl 2. The reaction was quenched by the addition of Tris base to a final concentration of 0.1 M. EDTA to a final concentration of 25 mM, and E. coli tRNA (prepared by the method of von Ehrenstein, G., Methods in Enzymology,

SP Colowick and NO Kaplan, Eds., Vol. 12A, p. 588 (1967)) 40 μg / ml. The reaction mixture was extracted with phenol, chromatographed on Sephadex G-100 and intermediate volume

2 the eluted Ρ-cDNA was precipitated with ethanol. This treatment yielded, in good yield, cDNAs with base pairs needed to attach chemically synthesized decanucleotides to the blunt end.

Hind III decamers were prepared by the following method: Scheller, RH Dickerson, RE, Boyer, HW,

Riggs, AD and Itakura, K., Science 196, 177 (1977).

The incorporation of Hind III decamers into the cDNA was performed by incubation for 1 hour at 14 ° C in a solution of 66 mM Tris-HCl, pH 7.6, 6.6 mM MgCl₂, 1 mM ATP, 10 mM dithiothreitol, 3 mM Hind III decamer with activity of 10⁵ beats per minute per picomole, and about 500 units / ml T4 DNA ligase. The reaction mixture was then heated at 65 ° C for 5 minutes to inactivate the ligase. It was then treated for 2 hours at 37 ° C with 150 units / ml Hsu I or Hind III endonuclease and then potassium chloride was added to a final concentration of 50 mM, γ-mercaptoethanol to a final concentration of 1 mM, and EDTA to a final concentration of 0.1 mM New England Bio-Labs, Beverly, Massachusetts, sells Hind III and Hae III endonucleases. The reaction product was analyzed by gel electrophoresis as in Example 1 and a peak corresponding to a sequence of about 450 nucleotides was observed, as well as truncated fragments of Hind III decamers.

Example 3a

Eco-RI decanucleotide recognition sites were ligated to the cDNA of Example 2 as described in Example 3. (Eco-RI decamers were prepared according to the aforementioned article by Scheller et al. And had the sequence 5'-CCGAATTCGG-3 '). The product was then digested with Eco R1 using the same reaction conditions as above (see digestion with Hsu I and Hind III). Eco R1 was obtained from New England Prolabs. The reaction product was analyzed by gel electrophoresis (as described in Example 1), and a peak corresponding to a sequence of about 450 nucleotides was found, as well as fragments of Eco-RI decamers.

Example 4

In this example, a recombinant plasmid is generated and characterized after replication has occurred.

Plasmid pMB-9 DNA (method of preparation: Rodriguez, RL, Boliver, F., Goodman, HM, Boyer, HW and Betlach, M., ICN-UCLA Symposium on Molecular and Cellular Biology,

DP Wierlich, WJ Rutter, and CF Fox, Eds., (Academic Press, New York 1976), p. 471-477) was cleaved from the Hind III restriction site by Hsu I endonuclease and then treated with alkaline phosphatase BAPF (Worthington Biochemical Corporation, Freehold, New Jersey). The reaction mixture contained 0.1 units of enzyme per 1 ug of DNA and was incubated in 25 mM Tris-HCl, pH 8, at 65 ° C for 30 minutes, after which the phosphatase was removed by phenol extraction. After ethanol precipitation, phosphatase-treated plasmid DNA was added to cDNA with Hind III cohesive ends at a ratio of 3 moles of plasmid per 1 mole of cDNA. The mixture was incubated for 1 hour at 14 ° C in a solution of 66 mM Tris, pH 7.6, 6.6 mM MgCl 2, 10 mM dithiothreitol, 1 mM ATP and 50 units / ml T4 DNA ligase.

The mixture was added directly to an E. coli X-1776 cell suspension prepared for transformation as follows. Cells were grown at 37 ° C to a cell density of g

about 2 x 10 cells / ml in 50 ml of nutrient solution containing 10 g / l tryptone, 5 g / l yeast extract, 10 g / l sodium chloride, 2 mM sodium hydroxide, 100 ug / ml diaminopimelic acid and 40 ug / ml thymine . Cells were harvested by centrifugation at 5,000 rpm for 5 minutes at 5 ° C, suspended in 20 ml of cold 10 mM sodium chloride solution, centrifuged as above, and resuspended in transformation buffer containing 75 mM CaCl 2, 140 mM NaCl, and 10 mM Tris, pH 7.5, and kept on ice for 5 minutes. The cells were then centrifuged and resuspended in 0.5 ml of transformation buffer. Transformation was performed by mixing 100 μl of cell suspension and 50 μl of recombinant DNA (1 μg / ml). The mixture was first incubated for 15 minutes at 0 ° C, then for 4 minutes at 25 ° C and finally for 30 minutes at 0 ° C. The cells were then transferred to agar plates for growth under selected conditions.

Screening of the recombinant plasmids was performed in the presence of 5 ug / ml tetracycline for Hind III site transformation. A specific recombinant, called PAU-1, was isolated. Impure plasmid preparations with 2-5 ug of DNA isolated from PAU-1 were treated with an excess of Hsu'I endonuclease. EDTA-Na was added₂to a final concentration of 10 mM and sucrose to a final concentration of 10% (w / v) and the mixture was separated on a polyacrylamide gel (8%). DNA was found at a site corresponding to a length of approximately 410 base pairs.

Example 4a. (i) Example 4 was repeated except that plasmid pMB-9 DNA was replaced with plasmid pBR-322 DNA prepared by Bolivar et al. as described (Gene 2 (1977) 95). The conditions used were otherwise the same, except that the final selection of recombinant clones was performed by culturing them in a medium containing 20 μg / ml tetracycline. The DNA sequence transferred to the recombinant clone was separated as previously described to give a DNA fragment of about 410 base pairs, the nucleotide sequence of which is shown in Table 1.

(ii) Example 4 was repeated except that plasmid pMB-9 DNA was replaced with plasmid pBR313 DNA prepared by Bolivar et al. by the method described (Gene 2 (1977) 75). The conditions used were otherwise the same except that the final selection of recombinant clones was performed as described in (i) to give a DNA fragment of about 410 base pairs, the nucleotide sequence of which is shown in Table 1.

(iii) Example 4 was repeated except that plasmid pMB-9 DNA was replaced with plasmid pSCIOI DNA prepared by Cohen et al. by the method described in (Proc. Nat. Acad. Sci.

USA 70 (1973) 1293). The conditions used (including the method for selecting recombinant clones) were as in Example 4. The DNA sequence transferred to the recombinant clone was separated to give a DNA fragment of about 410 base pairs, the nucleotide sequence of which is shown in Table 1.

Example 4b. Examples 4 and 4a were repeated except that

E. coli RRI or E. coli HB101 was used instead of E. coli X-1776. The conditions used were the same as in Examples 4 and 4a, and the same results were obtained.

Example 5

The plasmid PAU-1 DNA of Example 4 was further purified by electrophoresis on a 6% polyacrylamide gel. The DNA was then eluted from the gel

2 and labeled by incubation with P-ATP and polynucleotide kinase under the conditions described by Maxam and Gilbert fks. Proc.Natl.Acad.Sei., USA, 74 (1977) pages 560-). The enzyme catalyzes the transfer of the radioactive phosphate group f¹ 'P-ATP to the 5' ends of DNA. The enzyme was obtained from E. coli by the following method: Panet, A., et al., Biochemistry 12, 5045 (1973). The DNA thus labeled was digested with Hae III endonuclease as described in Example 2, and both fragments, one of about 265 base pairs and the other of about 135 base pairs, were separated on a polyacrylamide gel as described in Example 1. The isolated fragments were subjected to specific cleavage reactions and sequenced by the Maxam and Gilbert method mentioned above. Table 1 shows

4 6 4 0 a combination of the results of this series of experiments and the results of similar series of experiments in which cDNA was examined using plasmid vectors derived from Col E1, such as pMB-9 and pBR-322. The 5 'end of the sequence has an undefined sequence of about 50-120 nucleotides, and the length of the poly-dA at the 3' end varies. The sequence is based on the best current knowledge The corresponding amino acid sequence of rat proinsulin I starts at the triplet site numbered 1 and ends at the triplet site numbered 86. The sequence marked with a dashed line is still slightly uncertain.

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<td>d</td><td>o</td><td>tn</td>

<td></td><td>B</td><td> 44</td>

<td>B</td><td rowspan="2">O ί-</td><td> &lt;1&gt;</td>

<td></td><td>(Λ</td>

<td>d</td><td>ο</td><td></td>

<td>o</td><td>o o</td><td> 0</td>

<td>ί-</td><td>o</td><td> 4-'</td>

<td>ο</td><td> &lt;·</td><td>m</td>

<td></td><td>d</td><td>•B</td>

<td>B</td><td>o F '</td><td> 0</td>

<td></td><td></td><td> 4-&gt;</td>

<td></td><td>d</td><td></td>

<td>O</td><td>o</td><td> 4·'</td>

<td>o</td><td>o o</td><td>Tl &gt;</td>

<td>B</td><td>B Hl</td><td> 0</td>

<td>O</td><td> &lt;1 ·</td><td></td>

<td>O</td><td> &lt;1</td><td> 4&lt;</td>

<td rowspan="2">O</td><td>OH</td><td>O</td>

<td>Hl -1 * 1</td><td>Q)</td>

&lt;(

<td>O</td><td></td><td>Hl Hl</td><td>n3</td>

<td>d</td><td></td><td>OH</td><td> 4 &gt;</td>

<td>o</td><td></td><td>• rt</td><td> 3</td>

<td>CJ</td><td></td><td>O</td><td>ί- *</td>

<td>o</td><td></td><td>B</td><td>nj</td>

<td></td><td></td><td></td><td> &gt;</td>

<td>o</td><td></td><td>O</td><td>'"B</td>

<td>o</td><td>vO</td><td> * *</td><td>•B</td>

<td>o</td><td>C/O</td><td></td><td></td>

<td></td><td></td><td></td><td> &gt;</td>

<td>B</td><td></td><td>O</td><td>Φ</td>

<td>B</td><td></td><td>O</td><td></td>

<td>O</td><td></td><td>H *</td><td>r-4</td>

<td>O</td><td></td><td>O</td><td> &lt;</td>

<td></td><td></td><td>-c</td><td></td>

<td>O</td><td></td><td>B</td><td></td>

<td>O</td><td></td><td>O</td><td></td>

<td>ί-</td><td></td><td> &lt;</td><td></td>

<td>ο</td><td></td><td></td><td></td>

Example 6

In this example, the nucleotide sequence of human insulin is isolated, purified, and transferred into a plasmid essentially as described in Examples 1-4, starting from human pancreatic tissue obtained, e.g., from a donated pancreas or a freshly dead body or pancreatic islet tumor. Substantially as described in Example 4, a microorganism containing the nucleotide sequence code of the A chain and the B chain of human insulin is prepared. The known amino acid sequence of the human insulin A chain is as follows:

10 Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu- Tyr20 ·

Glu-Leu-Glu-Asn-Tyr-Cys-Asn

The known amino acid sequence of the human insulin B chain is as follows

10 Phe-Val-Asn-Glu-Kis-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu20 30

Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-Pro-Lys-Thr The amino acid sequences are numbered from the end with the free amino group, cf. Smith, LF, Diabetes 21 (suppl. 2), 458 (1972).