Untitled record

The present invention relates to a process for obtaining a microorganism that contains the gene for insulin production.

Insulin was first isolated in 1922. currently; Insulin required⁵as medicine or for scientific research is obtained by extraction from the pancreas, there are a large number of techniques for this purpose, given the need to achieve the highest yields and in 'în advantageous conditions. Although the slaughterhouses provide beef and pork straight pancreas_; sources of insulin, on a global scale, are more and more absent, the number of diabetics increasing. In addition, some diabetics have an allergic reaction to pork or beef insulin, with unpleasant, even harmful effects. Therefore, the question arises of finding new solutions to produce human insulin in sufficient quantities to meet the world's needs. These solutions need to make insulin out of animal organisms and in reasonable quantities. In some cases, for obtaining proteins, it was possible to obtain adequate cell lines; which can be maintained by tissue culture techniques. However, growth; the cells in tissue cultures are slow, the environment is expensive, the conditions must be controlled precisely, and the yields are reduced. In addition, it is often difficult to maintain a cultured cell line, which has the desired differentiation characteristics.

On the other hand, microorganisms have been found to obtain proteins, which, in certain culture media, with bitter substrates, can produce proteins.

Modern biological research has shown that in living organisms, from the simplest to the most complex, the synthesis and functioning of living matter is done according to a genetic program, contained in genes. The gene is a segment of the dc DNA or RNA macromolecule (in the case of riboviruses), consisting of a certain sequence of codons and nuclcotides, respectively, which function as a functional unit and contains the necessary genetic information.

LEI PRICE 59.74 synthesis of a polypeptide chain or other molecule.

In the current stage, the knowledge of the molecular structure of the genes and their functioning has been realized, which made possible their isolation as well as their artificial synthesis, thus allowing interventions in the structure and function of the genes, increasing the premises of isolation and transfer of genes to a microorganism. to other microorganisms (Prof. dr. P. Raicu, Biology, Bucharest 1978,

р. 72).

The present invention provides a process for obtaining a microorganism having a nucleotide sequence with the code for insulin, consisting of isolating cells from animal islands containing the mRNA code, for insulin from the pancreas of an organism, producing insulin, extracting mRNA from these cells. in the presence of an RNase inhibitory composition in order not to degrade mRNA, purification of mRNA to be released by protein, DNA or other RNA, DNA synthesis - double stranded, wherein a chain has a nucleotide sequence * complementary to that of mRNA, i.e., a cDNA, with a nucleotide sequence with the insulin code, preparing a DNA transfer vector with the reactant ends, capable of being linked together or capable of being double-stranded DNA binding, binding of the double-stranded DNA transfer vector, having a nucleotide sequence with the code for insulin, and finally mixing a microorganism with the DNA transfer vector bound with DNA, having a nucleotide sequence with the insulin code, wherein the mRNA extraction step containing the m code for insulin is performed by homogenizing the cells in the presence of an RNA-inhibitory composition, comprising 4 mM guanidinium thiocyanate and 0.05 mercaptoctanol. .. 1.0 mM at RH = 5.0, .. 8.0, and the step of performing the DNA transfer vector, with the reactive ends capable of being linked to one another or to be linked to a cDNA, having a nucleotide sequence for insulin, is performed by reacting cDNA with the transfer vector by enzymatic hydrolysis of a transfer vector selected from the group containing pM B-9, pBR-313, pBR-315. pBR-316, and pBR-322, with a restriction cndonuclease, selected from the group containing Hind III or Hsu I, preferably at RH == = 7.6 and temperature 37 ° C, for 2h.

6 examples of embodiment of the invention are given below.

Example 1. a) Description of microorganisms

Plasmid pBR-315 is described in the literature; the synthesis and characterization of this plasmid are found in the work of RL, Rodriguex and co-authors; over. pBR-315 carries a resistance gene against ampicillin dc (Ap^R) and a tetracycline resistance gene (Tc^R}. This plasmid contains a single resistance state for each of the Hind III, Sal I and Bam HI nucleases within the Tc gene.^K. pBR-315 also contains a single Eco RI restriction state and has a Pst I state within the Ap gene.^R. By inserting a foreign DNA into any of the Hind III states, S'al I or III loses Tc^R .

Thus, the recombinant molecules are found by selection according to the effects of Ap^R , Tc^s . The transfer vector can be characterized by the resistance properties against the drug, as well as by the restriction plane of the transfer vector, by the nucleotide sequence and the molecular weight of the introduced DNA, as described above.

RL Rodriguegue and co-authors have. also characterized by plasmid pBR-316. This plasmid carries an Ap gene^R and a Tc gene^R , pBR-316 has a single restriction state for each of the Hind III, Sal I or Bam HI nucleases within the Tc gene.^R . And pBR-316 has a Psf I restriction state within the Ap gene^R and does not contain Eco RI status. By inserting a foreign DNA into one of Hind 111, Sal I or Bam HI, Tc is lost^R . Accordingly, the recombinant molecules are found by selection for the effects of Ap^{! i} and Tc^R. The transfer vector may be characterized by the drug's resistance properties, as well as by the restriction plane of the transfer vector and then by the nucleotide sequence and molecular weight of the introduced DNA, as described above.

E. coli X-1776 is a strain of E. coli R 12 developed by Ray Curtiss and described in Ann. Iiev. Microbiol. 30,507 (1976), as well as in Patent, USA, no. 4 190495. E. coli X-1776 sc is stored in the American Type Cnlture, at no. 3 244 E. coli X-1776 sc characterizes as Ftau A53 dap D8 merAl supE42 Δ40 (galuvrB) - minB2 malA25 thv A57 metC65 Δ29 (bioH-asd) cysB2 cyclic HsdR2.

E. coli RR, is a strain of E. coli K12 described by F. Bolivar and co-authors in Gene 2.95 (1977). li. RR1 sheets are characterized as F pro Iue thi Iac Y Str rPiniă.

Plasmid pRI-1 comprises pBR-322, having a nucleotide sequence, which encodes rat insulin inserted therein. pRI-1 is kept in the collection A merican Type Cnlture (d ICC), number 40003. £. coli RRj / pRI-1 is E. coli RRI containing plasmid pRI-1.

The new feature of this microorganism is that it contains a plasmid that bears

782C0 the nucleotide sequence for rat dc insulin. E. coli RRI / pRI-1 is maintained in. ATCC at · no. 392. The RGH plasmid, having a nucleotide sequence inserted in it, encodes the entire rat growth hormone. The new feature of this plasmid is that. contains the nucleotide sequence: whole, for rat growth hormone. The RGH plasmid is stored in the ATCC, at no. 40 001. E. coli X 1776 / RGH is E. coli X-1776, which contains the RGH plasmid. The new feature of this microorganism is that it contains the plasmid that carries the nucleotide sequence for the rat growth hormone. E. coli X-1776 is kept in the ATCC at no. 3.1390.

Plasmid pBRr322 is extensively characterized. So. F. Bolivar et al. in the. Gene 2, 95 (1977) describes the synthesis and characterization of this, special plasmids as having a molecular weight of 2.7 x IC⁶ daltons (revised by Bolivar, F. in Gene 4, 12.1 (1978), and carries a gene for ampicillin resistance (Ap^R and a tetracycline resistance gene (Tc^R).

in the gene Ap^R there is only one restriction position for Hind III, Sal I and Bani-HI endonucleases. Also, pBR 322 contains a single Eco RI restriction position, two Hinc II restriction positions. five Eco R II restriction positions, three Bgl I restriction positions 12 Alu I restriction positions, twelve Hae II restriction positions and seventeen Hac III restriction positions. Map of the circular restriction for pBR. 322: it is included on page 102, from the work of Bolivar et al. A?^Kit is lost if. pBR 322 splits into position Pst. I and: DNA is introduced. · Similar; you lose tc^R if pBR 322 is cleaved with Hind III or Sal I or Bani HI and DNA is introduced. Introducing DNA into. position.Pst I, the recombinant carcass molecules find, selecting, the colonies sensitive to. ampicillin (Ap^s) and Tc^K. Similarly, by inserting DNA into one of the Hind III, Sal I, or Bam HI positions, the recombinant molecules are selected. the colonies that are Ap^Rand Tc^s·. A vector of. transfer, which contains foreign DNA: introduced into any of the four positions, discussed above, can be characterized, in terms of the drug resistance properties of the transfer vector.^s Tc^K or Ap^E'Tc^of. The transfer vector can be characterized: further by. introduced DNA removal and: determination of the molecular weight of the two products compared with the molecular weights of the materials from: to. who to. started. Another characterization see can- realized by making, α map of: constraint. ' a. vector: transfer. The DNA sequence of the insert can also be determined.

Similarly, plasmid pBR 313. was also characterized. The synthesis and characterization of this plasmid is described by

F. Bolivar et al. Gene 2 75 (1977). pBR 313 has a molecular weight of 5.8 XX 10^B daltons and carries a gene for ampicillin resistance (Ap^R) and a gene for resistance to tetracycline (Te). pBR-313 contains a single restriction position for each of the Hind III, Sal I and Bam'iAȚ nucleases, within the Tc gene.^R . An exhaustive analysis of the restriction endonuclease of pBR-313 was made, making up the restriction map, which can be found on page 84 of the article by Bolivar et al. Introducing foreign DNA into one of the positions of Hind III, Sal I or Bam HI, Tc^R is lost. Thus, the molecules that recombine, are found by selecting the strains for Ap^R , Tc^s. The transfer vector can be characterized by the drug resistance properties, as well as by the DNA restriction map sequence introduced and by. molecular weights, as described above.

A third plasmid that has been extensively characterized is pMB-9. This plasmid is prepared in the manner described by RL Rodriguez et al. and by F. Bolivar et al. Gene 2, 75 (1977). pMB-9 has a molecular weight of 3.5 χ 10 daltons and carries a tetracycline resistance (Tc) gene.^R). This plasmid has a single restriction position for each of the Eco RI, Hind III, Sal I or Bam HI endonucleases. These three latter positions are located within the Tc gene^R. If you enter in any of the · Hind III, Sal I or Bam HI positions, Tc is lost. A transfer vector containing a foreign DNA in one of these positions can be characterized from the point of view of this property. This transfer vector can be further characterized by analyzing the DNA sequence of the insert, comparing the molecular weights and analyzing the restriction, as described above.

The pSC-101 plasmid was also extensively characterized. Thus, S. ii. Cohen and others in Proc. Nat. Acad. Sci. USA, 70, 1293 (1973), describes the synthesis and initial characterization of this plasmid. Further characterization can be found in SN. Cohen et al. in Proc. Nat. Acad. Sci USA, 70, 3240 (1973) at HW Bayer et al, in Recombining Molecules, RF Beers and Basset, EG Eds; Raven Press, New York, p. 13 (1977) and GN Cohen et al., In Recombining Molecules, 8, p. 91. pSC101 has a molecular weight of 5.8 x χ 10⁶ daltons and carries a gene for tetracycline resistance (Tc^R). within the gene there is a single restriction position each for the endonucleases HIW.IIi,

Sul I and Bam HI. In addition, pSclOl contains a single EcRI restriction position, a single APal position, a single Ema position 1 and four Hinc II positions. Tc^K it is lost if pSC-101 splits with Iiind III or Sal I or Bam HI and ADR is introduced. By inserting DNA into any of the Hind III, Sal I or Bam I positions, recombinant molecules are found by selecting colonies that are Tc.^it . A transfer vector containing foreign DNA, inserted within any of these three positions, can be characterized from the point of view of the drug resistance property of the transfer vector. The transfer vector can be characterized by:

(a) - removal of waxed DNA and determination and comparison of molecular weights;

(b) - drawing up a restriction map;

(c) - determining the DNA sequence a. insertion, as discussed above.

The phages used for transfer vectors comprise a class of lambda derivatives of phage called Charon, in particular Charon 3A, Charon 4A and Charon 16A. These phages have been extensively characterized. Thus, Blatner et al. describe the synthesis and characterization of these phages. On p. 161 of this paper a physical map of these phages is found, including the positions of the restriction endonuclease. The genotype of each phage is indicated on p, 164, with the results of cleavage by a given restriction endonuclease and the dc method, if a foreign DNA has been successfully reproduced in the phage. For example, by inserting a foreign DNA into the EcoRI position of a Charon 16A, the Lac5 gene is lost, the growth of recombinant phage on the Lac lac bacteria results in colorless plaques. Thus, the transfer vector containing foreign DNA introduced into a suitable position can be characterized by the genetic properties of the transfer vector, for example colorless plaques on Lac bacteria. The transfer vector can be further characterized by removing the introduced DNA and determining the molecular weights of the two products, compared to the molecular weight of the initial elements. Further, the characterization can be made by drawing a restriction map of the transfer vector. It can also deteriorate. mine and DNA sequence of the insert.

Similarly, WESλΒ is well characterized by a bacteriophage derivative of lambda. The synthesis and initial characterization of this phage are described by 1). Tremaier et al. in Nature, 263, 526 (1976). The additional features of this phage can be found in P. Leder et al. Science., 196, 175 (1977). The genotype of this phage is identified in two beautiful references. The last article further identifies the location of the two restriction positions for Eco Rl and the two positions for Sst I. The beech contains four positions for Bam HI, giving fragments of 5.4 χ 10^{: s}; 19.3 χ IO³; 3.8 χ.10³ and 11.4 χ 10^s base pairs in length. The analysis by restriction endonucleases is shown on page 527 of the article by Tremaier et al. Fragment 1B is removed by digestion with Eco R1 and split by Sst I. This prevents recombination with fragment XB. The foreign DNA, having the ItcoRI links, sc introduces in the beech in the Eco Rl positions. Phages are screened for in situ hybridization recombination clones, as described by WD Benton and RW Davin, in Science, 196, 180 (1977). The transfer vector can be characterized:

(a) - by removing introduced DNA and by determining and comparing molecular weights, (b) - by restriction endonuclease analysis;

(c) - by determining the DNA sequence of the insert, as discussed above.

b) Extraction and isolation of AUXm from rat insulin; synthesis of complementary DNA and characterization of complementary DNA

In order to prepare purified cells from rat islands, a solution of Hanii salt was injected into the pancreas of an anesthetized rat by retrograde injection into the pancreatic tube. The Hank salt solution is a standard mix of known saline solution (Grand Island Biological- Supply Company, Grand Island, New York, USA). The pancreas was then removed, chopped in Hank's solution at 0 ° C and soaked with eolagenase and trypsin inhibitor from soy beans. All operations were performed at temperatures of O ... 4 ° C. The conditions of the digestion operation were extremely critical. Two rat pancreas minced in Hank medium in a total volume of 8 ml, were placed in a 30 ml glass tube. All glass tubes were treated with silicones. The incubation mixture contained 12 mg of eolagenase (an enzyme prepared from Closlridium hislolyticmn) by a literature method (Mandl I. et al., Journal of Clinic InvestigalionEl, 1943 p. 1323), type CLS IV (Worthingion Biochemical Corporation Ereeland, New Jersey USA) and 1 mg soybean trypsin inhibitor. Incubation was performed at 37 ° C for 25 minutes, with a shake of 90 times per minute. Continuous inspection was required to ensure that collagen activity was optimally performed. If the incubation is too short, the cells in the islands are not completely free, and if the incubation is too long, the cells in the islands begin to break. After incubation, the tube was centrifuged for min., At 200 χ G. The supernatant was decanted and the pellet washed with Hank's solution, this operation being repeated five times. After final centrifugation, the pellet was suspended in 15 ml Eicoll (Ficoll trademark - Pharmacia Chemical. Company, Uppsala) product, t-density, 085. An 8 ml layer of Ficoll product of density 1,080 was added, one layer of; 5 tul Ficoll product of density 1,060 and the tube was centrifuged in a rotor with oscillating cups for 5 min, at 500 χ X. G, then for another 5 min, at

000 χ G. As a result of this operation, at. the bottom of the tube remained the cells of action, and the cells of the islands climbed into the density gradient and formed a bank between the two upper layers. The cell strip of the islands contained contaminating ganglion cells, lymph nodes and connective tissue. Large contaminant fragments were removed from the material. band. The rest of the preparation was placed under a dissecting microscope, where the visible contaminating material was removed by hand, using a micropipette. After that, the cell preparation was diluted in Hank's solution and centrifuged. The supernatant was decanted and the cell pellet was stored frozen in liquid nitrogen.

Island cells collected from 200 rats were homogenized at 4 ° C in 4 M guanidinium thiocyanate containing betamercaptoethanol buffered at pH = 5.0. The homogenate was poured over 1.2 ml 5.7M CsCl containing 100 mM EDTA and centrifuged for 18 h, at 37,000 rpm in a SW 50. 1 centrifuge rotor; RNA went to the bottom of the tube.

From the total RNA preparation, it was isolated by. chromatography on oligo (dT) - cellulose. according to a known method in the literature (Aviv II, Lcder P, Proc Natl, Acad. Sci. USA 68, 1972 p. 1408) polyadenylated RNA.

For total polyadenylated RNA transcription from rat Langerhans islands to DNA, reverse transcriptase from bird myeloblastosis virus (Dr. DJ Beard, Life Sciences Incorporated, St. Petershurg, Florida, USA). The reactions were carried out in 50 mM Tris-HCl, pH = 8.3, 9 mM M Mg Cl₂, 30 nM NaCl. 20 nM iefa-mercaptoethanol, 1 mM of each of the 3 non-radioactive deoxyribonucleoside triphosphates, 250 μM of the fourth alpha-labeled deoxynucleoside triphosphate ~³² P, of specific activity 50,. „.200 Curie / mol, 20pg / ml oligo-dT_{13 ] S}commercially (38), 100 pg / ml polyadenylated RNA and 200 units / ml reverse transcriptase. The mixture was incubated at 45 ° C for 15 min. After addition of EDTA.-Na ^ at 25 mN, the solution was extracted with an equal volume of water-saturated phenol, followed by aqueous phase chromatography on a Sephadex G-100 column, 0.3 cm in diameter and 10 cm high. , in 10 mM Tris-HCl, at pH = 9.0, 100 mM NaCl, 2 mM EDTA. The nucleic acid, glued to the free volume, precipitated ch-, ethanol after addition of ammonium acetate UppH - 6.0, ... 0.25 M, The precipitate was collected by centrifugation, the pellet dissolved in 50 μl solution of NaOH 0, IN freshly prepared and incubated at 70 ° C for 20 min to hydrolyze RNA. The mixture was neutralized by the addition of 1N sodium acetate, pH = = 4.5, and the product 32P-cDNA, precipitated with ethanol and redissolved in water. Aliquots of single-stranded cDNA were analyzed on native polyacrylamide gels by a method described in the literature (38). Gels were dried and 32 P-DNAs were detected by autoradiography using a commercial film (39). CDNA was lieterodispersed, judging by the electrophoretic model. It contained at least one prominent cDNA species, with approximately 450 nucleotides, judging by comparison with known standards.

Example at. The above example is repeated, but the β concentration varies. of mercaptoethanol in the homogenization stage of island cells. The concentrations of âefc-mercaptoethanol that are tested are: 0.05 M, 0.2 M, 0.6 M and 0.8 M. All concentrations of dc feefc-mercaptoethanol, which were used, gave identical results, respectively. mRNA degradation by NRase is prevented.

Example lb. Repeat Examples 1 and 1a, but sc varies the pH of guanidine thiocyanate and Z> r «-mercaptoethanol; In the homogenization stage of the island cells the tested ρΗs are: 6.0, 7.0 and 8.0. At all the pHs examined, identical results were obtained, ie the mRNA degradation by RNase is prevented.

Example 2. We describe the synthesis and characterization of double-stranded cDNA containing the rat insulin sequence. The single-stranded cDNA product of Example 1 was treated with reverse transcriptase to synthesize the complementary chain. The reaction mixture contained 50 mM Tris-HCl; pH = 8.3; 9 'mM Mg Ch, 10 mM dithiothreitol; 50 mM of each of the unlabeled deoxyribonucleoside triphosphates, 1 strand of a nucleoside triphosphate labeled with alpha-32 P of specific activity 1 ... 10 Cnrie / thousand, 50 g / ml cDNA and 220 units / / ml reverse transcriptase. The reaction mixture was incubated at 45⁰ C, for

120 min. The reaction was stopped by the addition of EDTA-Na, at 25 mM, phenol extraction and chromatography on Sephadex G-100, followed by precipitation with ethanol. An aliquot part of the reaction product, having 500, 1000 cpm, was analyzed by gel electrophoresis, as described in Example 1. A heterodisperse band was observed, averaging approximately 450 nucleotides in length, determined by comparison with standard samples. Aliquots of the DNA reaction products of Example 1 and Example 2 were treated separately by digestion with excess restriction endonuclease Hae III and ana> _t >

similarly lysed by gel electrophoresis. Both products were deactivated by endonucleases, so two bands of radioactivity were observed at gel electrophoresis. The bands resulting from the double-stranded cDNA cleavage essentially represented cleavage products of the same length as those resulting from the single-stranded cDNA cleavage.

Example 3. Describe the binding of the truncated ends of the Hind III decanucleotide linkages to the double-stranded cDNA in the rat islands of Example 2. The double-stranded reaction product of Example 2 is treated at a concentration of 2 ... ... 5 pg / ml, with 30 units of SI nuclease, having an activity of 1,200 units / ml, obtained commercially, in 0.03 M sodium acetate, / TI = 4.6, 0.3 M sodium chloride, 4 , 5 mM Zn.CE, at 22 ° C, for 30 min of incubation, followed by another 15 min of incubation, at 10 ° C. To stop digestion, the addition of Tris base to a final concentration of 0.1 M, EDTA at 25 mM and E. coli tRNA, prepared according to a literature method and at 40 µg / ml, is used. After phenol extraction of the reaction mixture and chromatography on Sephadex G-100, 32 P-cDNA eluted in the free space was precipitated with ethanol. This treatment resulted in a high yield of cDNA molecules, with paired base ends, required for ligation of truncated ends to chemically synthesized decanucleotides. Hind III decoctions were prepared using a method described in the literature. The ligation of Hind III decamers to cDNA was performed by incubation at 14^CC ·, for one hour, in 66 mM Tris-HCl, p = 7.6; 66, mM MgCh; 1 mM ATP; 10 mM dithiothitrcitol; 3 mM Hind III decamers having 10⁵ cpm / / mol and T4 DNA ligase, approximately 500 units / ml.

The reaction mixture was then heated to 65 ° C for 5 nrin to inactivate the ligase. KC1 was added to 50 mM final concentration, ftcZu-mercaptoethanol, 1 mM final concentration and EDTA to 0.1 mM final concentration, before su.12 digestion with 150 units / ml endonuclear Hat I or Hind III time from 2h, at 37 ° C. Hind III or Hme III endonucleases are commercially available. The reaction product was analyzed by gel electrophoresis as in Example 1 and a little was observed, corresponding to a sequence of about 450 nucleotides, along fragments of cleaved Hind III decamers.

Example 3a. The Eco-RI decanucleotide bonds are bound by a DNA-stranded end of Example 2, as described in Example 3. ECORI'S Depamer prepares as described / Schneller et al. and have the sequence 5 LCCGÂATTCGG-3 '. After hollow end binding, the product is digested with ECORI, using the same reaction conditions as those described for Hsu I or Hind. III. ECORI sc can be found in trade delivered by Biolaboratoarcle from New England. The reaction product is analyzed by gel electrophoresis, as in Example 1, with a peak corresponding to a sequence of approximately 450 nucleotides, in addition to the fragments of the cleaved Eco RI decamers.

Example 4. Sc describes the formation of a recombinant plasmid and its characterization after replication. A pHB-9 DNA plasmid, prepared according to a method described in the literature, is cleaved into the Hind III restriction position with Hsu I endonuclease, then treated with commercially obtained alkaline phosphatase BAPP. The enzyme is present in the reaction mixture at the · level of 0.1 units / pg DNA and the reaction mixture was incubated in 25 mM Tris-HCl; pH = 8; for 30 min, at 65 ° C, followed by extraction with phenol for phosphatase removal. After ethanol precipitation, the phosphatase-treated DNA plasmid was added to the cDNA containing Hind-III cohesive terminal groups in a molar ratio of 3 mol plasmid to 1 mol cDNA. The mixture was incubated for one hour in 65 m Tris; / TT = - 7.6; 6.6 mM MgCh; 0 mM dithiothreitol and 1 mM ATP at 14 ° C; in the presence of 50 units / ml of T4 DNA ligase.

The ligand mixture was added directly to an E. coli X-1776 cell suspension, prepared for transformation as follows: the cells were grown at 37 ° C up to a cell density of 2 χ 10⁸ cells / ml in 50 ml medium containing 10 g / l Tryptone, 5 g / l yeast extract, 10 g / l NaCl, 2 mM NaOH, 100 g / ml ml diaminopimalic acid and 40 µg / ml thymine. The cells were harvested by centrifugation for 5 min at 5 000 χ C and 5^UC, resuspended in 20 ml cold solution, 10 mM NaCl, centrifuged as before and resuspended in 20 ml transformation buffer containing 75 mAI

7S200

CaCȚ, HO mM NaCl and 10 mM Tris, /> H = ---- 7.5, then left for 5 min in ice. After that, the cells were quenched and resuspended in 0.5 ml of transformation buffer. Transformation was performed by mixing 100 µl of the cell suspension with 50 μΐ DNA for recombination (1 µg / ml). The mixture was incubated at 0 ° C for 15 min, then shifted to 25 ° C. for 4 min, then at 0 ° C, for 30 min. The cells were then transferred onto agar-agar plates for growth under selection conditions.

The separation, to obtain the recombinant plasmids, was performed at 5 µg / ml tetracycline for conversion to the Hind III position. A selected recombinant, designated with pAU-1, was isolated. Crude plasmid preparations of 2 µg - µg DNA isolated from pA.U-1 were digested with an excess of Hau 1 enconucleases. Then 10 mM EDTA-Na was added_of and sucrose 10% by weight final volume concentration and the mixture was separated on 8% polyacrylamide gel, the DNA was found at an appropriate position at about 410 base pairs in length. In a similar experience, plasmid pBR-322 was used as a transfer vector. All conditions were as described, except that the final selection of the recombinant helens was performed on plates containing 20 µg / ml ampicillin,

Example 4a. (i) Repeat example 4, using plasmid pBR-322, which prepares as described in Bolivar et al in Gene 2, 95 (1977) instead of plasmid pMB-6 DNA. All conditions are described except that a final selection of the clones that are recombined for growth on plates containing 20 g / nil tetracycline is made. The insert is removed as described, at. obtaining a DNA of about 410 base pairs in length and having the sequence shown in table 1.

(ii) Repeat Example 4 using plasmid pBR-313 DNA, prepared as described by Bolivar et al., Gene 2, 75 (1977) instead of plasmid pMB-9 DNA. All conditions are described, except for the final selection of the recombinant clones, as described in the DNA preparation of approximately 410 base pairs in length and having the sequence shown in Table 1.

(iii). Example 4 is repeated, using the plasmid pSCIOI DNA prepared according to the method described by Cohen et al., Proc. Nat. Acad. Sci USA, 70, 1293 (1973) instead of plasmid pMB-9 DNA. All conditions, including selection of recombinant clones, are described. Remove the insert as described in the DNA preparation of ι · about 410 base pairs in length and having the sequence shown in Table 1.

Example 4b. Sc repeats examples 4 and 4a in which E. coli RRI or E. coli HB 101 is used instead of E. coli Β X-1776. All conditions are described and identical results are obtained.

Example 4c. (i) Charon 16A DNA is prepared in the manner described by Blattner et al. and used AS a transfer vector. Sinf cohesive headsHeated by 60 ml incubation!) 42 ° C in 0.1 M Tris-HCl; H- ~ 8.0 and 10 mm MgCl₂. The vector is cleaved in the ECORI restriction position with ECORI endonucleases, then treated with an alkaline phosphatase, as described in Example 4. After precipitation with ethanol, the cDNA, treated with phosphatase, is added to the cDNA containing ECORI cohesive ends, in - a non molar ratio of 2 mol vector versus 1 mol cDNA. The spherical mixture bound to T4 DNA ligase, as described in Example 4. The binding mixture is added directly to an E-cell suspension. sheets X 1776 preparations for transformation, as described in Example 4 and transformation sc performs as in Example 4. Recombinant phages sc recover and are plated on Lac bacteria, on plates containing 5-chloro-4-bromo-3-indolyl i > ef «-D ~ glactoside (x 6), select 80, ug / ml recombinant phages containing cDNA introduced in Charon 16A ECORI position to produce colorless plates. A selected recombinant is isolated and digested with an excess of ECORI endonucleases and the resulting product is analyzed in the manner described in Examples 4 and 5. A DNA of approximately 401 base pairs in length is recovered and having the sequence shown in Table 1. Alternatively, the binding mixture is used to form recombinant phages by in vitro packing, as described by N. Sternberg et al., Gene 1, 255 (1977). Recombinant phages can be screened using the hybridization technique of in situ infection. described by WD Benton and RW Davis in Science 196, 180 (1977).

(ii) Charon DNA 3A prepared in the manner described by Blattner et al. is used as a transfer vector instead of the Charon 16A DNA used above. All conditions are identical to those described for Charon 16A DNA. A selection of recombinant phages is made by plating phage on Lac bacteria on plates containing X 6 and isolating colorless plates, followed by either hybridization to a suitable sample or digestion. of restriction endonuclease, as described by Blattner et al. The insert is removed in the manner described above when preparing a DNA of about 140 dc base lengths and having the sequence shown in Table 1.

(iii) gt WES DNA. B, prepared in the manner described by Ticmier et al, is used as a transfer vector instead of Charon 16 A, used above. All conditions are identical to those described for Charon 16 A. The selection of recombinant phages is performed by hybridization method of Benton and Davis. The insert is removed as described above, preparing an AI) X with approximately 140 base pairs in length and having the sequence shown in Table I.

Example 4d. Repeat Example 4c using E. coli RRI, E. coli HB 101, E. coli DP 50 or E. coli DP 50 Sup F instead of E. coli X-1776. All conditions are identical, the results obtained are also identical.

EXAMPLE 5 The DNA from pAU-1 described in Example 4 was further purified by electrophoresis on 6N polyacrylamide gel. After gel elution, the DNA was labeled by incubation with the 32 P-ATP range and the polynucleotide enzyme kinase, under the conditions described in the literature, the enzyme catalyzes the transfer of a radioactive phosphate group from the 32 P-ATP range to the 5 'ends of the DNA. The enzyme was obtained from E. coli by a method described in the literature. The DNA thus labeled was cleaved with endonuclease Hsc III as described in Example 2, and the two labeled fragments, approximately 265 and 135 base pairs respectively, were separated on a polyacrylamide gel, under the conditions described in Example 1. Fragments isolates were subjected to specific cleavage reactions and sequence analysis, by the method described in the literature. The sequence in the table below is based on an assembly of the results from the present experiments and those from a similar series of cDNAs, using plasmid vectors from Col. 1, such as pMB-9 and pMH-322. In the sequence at the 5 'end, a sequence estimated at 50 to 120 nucleotidc in length is indeterminate and the poly dA segment at the 3' end is of variable length. The sequence is given as representing the best information available today, in the sense that further studies may reveal additional details or indicate the need for easy revisions in some areas. The amino acid sequence corresponding to the proinsulin i in the rat begins at the triplet position marked with 1 and ends at the triplet position marked with 86. There remains some uncertainty about the sequence underlined by the dashed line.

Example 6. The separation and purification of DNA is described - having a nucleotide sequence encoded for human insulin and, at one time, the synthesis of a transfer vector containing that DNA and the construction of a microorganism strain that contains DNA as part of its genetic composition.

Human islet cells are isolated from human pancreatic tissue, as described in Example 1. Pancreatic tissue is obtained from a suitable human source, for example as a donated pancreas or from a fresh carcass or from a human insulin. The cells of the islets collected from several pancreas are homogenized in 4M thiocyanate, which contains 0.2 M fo / a-mccaptoethanol, the fold being 5.0, as described in Example I. Polyadenylated RNA sc isolates by analogue chromatography, as described by Aviv and Leder. The single-twisted cDNA was prepared using reverse transcriptase and hydrolyzed RNA, as described in Example 1. The double-twisted cDNA was prepared using reverse transcriptase, as described in Example 2, and digested with S1 nuclease as described in Example 3. Insulin human DNA binding agents III are added twofold DNAs, according to example 3. The product is digested with He ind III or HSU I and analyzed, as described in Example 3. A peak corresponding to a sequence of approximately 450 additional nucleotides is observed relative to the hindlimb release fragments. Human insulin cDNA containing cohesive ends binding to pMB-9 plasmid, as described in Example 4. E. coli X-1776 is transformed and screened for recombinant plasmids, as described in Example 4. The insert is removed with Hsu I and analyzed in the manner described¹.In Example 4. A DNA with about 450 nucleotidc is recovered. - Human insulin has been found to comprise nucleotides encoded for the entire amino acid sequence of human insulin. j.

The amino acid sequence of the A chain is:

'. 10 Gly-Ilc-Val-Glu-Gln-Cys-Cys-Thr-Ser-IleCy-Ser-Leu-Tvr-Elu-Eeu-GIu-Asu-Tyr- ',.

Gys-DNA.

The amino acid sequence of the B chain is:, '' 10

Phe \^zof-Asn-Glu-His-Leu-Cys-Gly-Ser10

His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val20. ··. .

Cys-Gly-Glu-Arg-Gly-Phe-Tyr-Thr-Pro

Lys-Thr.

The amino acid sequences are numbered at the end, having a free amino group.

Example 6a. (i) Repeat example 6 using plasmid pBR-322 in place

7S200 plasmid pMB-9 DNA. All conditions and selection of recombinant plasmids are identical to those described in Example 4 a (i), Insertion is removed as described, resulting in a DNA of approximately 450 nucleotides, having the sequence described in Example 4.

(iî) Repeat Example 6 using plasmid pBR-3; 13 DNA instead of plasmid pMB-9 DNA. All conditions and selection of recombinant plasmids are identical to those described in Example 4 a (ii). The insert is removed as described to obtain a DNA of about 450 nucleotides, having the sequence described in Example 6.

(iii) Repeat Example 6, using plasmid on DNA Scolium instead of plasmid pBBa DNA. All conditions and selection of recombinant chlorides are identical to those described in example 4â (iii). The insert is removed as described, obtaining a DNA with about 450 nucleotides and having the sequence described in Example 6.

Example 6b. Repeat examples 6 and 6a in which E. coli RR1 or E. coli HB 101 is used instead of E. coli Χ-1Π6. All conditions are as described, and the results obtained are identical.

Example 6c. Human insulin cDNA, prepared in Example 6, sc is treated by adding chemically synthesized ECORI bonds, as described in Example 3 a.

(i) Insulin · human DNA treated with ECORI is introduced into the ECORI position of Charon 16A, as described in Example 4c (i). The recombinant phages sc recover and screen, and the insert is removed as described in Example 4 c (i). A DNA is recovered with about 450 nucleotides and having the sequence described in Example 6.

(ii) The human insulin cDNA having cohesive ends ECORI is inserted into the Charon 3A vector as described in Example 4 c (ii). The recombinant phages are selected and analyzed as in example 4 c (ii), obtaining a DNA with about 450 nucleotides and having the sequence described in example 6.

The cold example. Repeat example 6c wherein E. coli RR], E. coli HB101, E coli DP 50 or E. coli DP50 Sup F is used instead of E. coli X-1776. All the conditions are the same as those described here and they obtain identical results.

AAC TAC TGC AAC TGA GTTCAA TCAA TTCCC G ATCCACCCCTCT GCA ATG AA TAAAGCCTTTGAATGAGC - poles A Marked sections = Domains of uncertainty at present.

Phe-Val-Asn- GlU-His- Leu-Cvs-Fly-Ser10

His-Leu-Val-Glu-Ala-Leu-Tyr20

Leu-Val-Cys- Gly- Glu-Arg- Gly-Pce-Phe30

Tyr-Thr-Pro-Lys-Thr.

The amino acid sequences are numbered from the end having a free amino group (Maxam AM, Gilbert W, Proc. Natl. Acad. Sci USA 74, 1977, ρ. 56θ /

The microorganism obtained with the gene of the upper organism has the code for human insulin, respectively contains a nucleotide sequence. Which encodes the chain A of the insulin «dwarves, ·· they contain: 5 '--- GGL, ATM₂ GT.1,_{, Q}. GAJ .. CAj ·, TGK ,, TGC₇ ACL «QR₀ETîATM₁₀ TGK _u QR_1SS ,, X ,, TY₁₃ TAK, gee₁₅ x₁₀ty ₁₆PLEDGE₁₇ AAK_1S TAK_1S TGK_z? AAK_n- ~ 3 'wherein A is deoxidenyl, G is deoxyguanil, C is deoxytosyl, T is thymidyl, J is A or G, K is T or C; L is A, TC or G; M is A, C or T; Nn is T or C, if Yn is A or G, and C if Yw is C or T; Y «is A, G, C or T, if Xw is C, and A or G if Xn is Τ; Wn is C or A, if Zm is G or A, and C if Zn is C or T; Zn is A, G, C or T, if W «is C, and A or if Wn is A; As for TC, if Sjj is A, G, C or T, and AG if Sw is T or C; S «is A, G, C or T, if QRw is TC, and T or C if QRu is AG; and the subscribed numbers, n, refer to the position of the amino acid in human insulin, to which the nucleotide sequence corresponds, according to the genetic code, the amino acid positions being numbered from the amino end, also contains an additional nucleotide sequence encoding the B chain of the insulin. human, comprising:

5 '- - -TTKj CTL₂ AAK₃ GAJ.CAK _sX _cTY₆TGK ₇ GGL _s QR₀S ₀CAK_1O X_u TY_U CTL₁₂PLEDGE ₁₃ GCL₁₄ X_ls TY₁₅ TAK_1CX ,, TY₁₇GTL i_STGK _1B GGL,₀ PLEDGE₂₁ W₃₃ CZ ,, GGL₂₃ TTK_2I TTK₂₅ TAKae ACL₃₇CCL₂₈AAJ_AFL ACL ₃₀--- 3 'in the microorganism, the nucleotide sequences encoding human A and .B chains of human insulin are joined by a nucleotide sequence (N «NZ> Nc) J in the 5' partial sequence --- ACL_A0 (NaN & Nc)

- - 3 ', where Να, N6, and Nc can be A, Ț, G or.C and j is an integer from 0 to 100, provided that NaNâNc is neither TAj nor TGA; the nucleotide sequence encoding the human insulin chain is carried by a DNA transfer vector, which is a plasmid selected from the group consisting of pMB-9 and pBR-322 (deposited in ATCC No. 40 0003 / PRI-1) and the microorganism is Escherichia coli, RR-1 / PRL - filed in ATCC collection no., 31 391 (American Cnltwrc. Collection, 12 301 Parklaron dr. Rockwille Maryland 20 852), and the nucleotide sequence encoding human B-chain B is carried by a DNA transfer vector, which is

7S200 a plasmid selected from the group consisting of pMB-9 and pBR-322 and the microorganism is Escherichia coli.

For a better understanding of the invention we show the following;

The sequence of steps of the process, according to the invention, can be classified into four general categories.

1. Isolation of a desired cell population from a higher organism

There are two potential sources of a genetic coding sequence for a specific protein; DNA from the source body itself and an RNA transcript of DNA. In the US, the current safety requirements of the National Institute for Health specify that human genes of any kind cannot be introduced into recombinant DNA and then into bacteria only after the genes have been purified very carefully or in special facilities (P₄) high risk (Federal Register vol. 41, no. 131, July 7, 1967, pp. 27 902 .... 27 943). Therefore, for any mode of work having the potential utility of producing human protein, as in the present process, the preferred access pathway is the isolation of a specific mRNA, having a nucleotide sequence encoding the desired protein. Adopting this strategy also has the advantage that mRNA can be purified more easily than DNA extracted from the cell. In particular, one can take advantage of the advantage that, in highly differentiated organisms such as vertebrates, it is possible to identify a specific cell population, having a specific location in the body and whose function is primarily devoted to the production of the protein in question. Alternatively, such a population may exist during a transient stage of development of the organism. In such cell populations, much of the mRNA isolated from the cells will have the desired nucleotide sequence. Therefore, the choice of the isolate cell population and the isolation method used may be largely advantageous in terms of the initial purity of the isolated mRNA from this population.

in most tissues, glands, and organs, cells are maintained together by a generally fibrous network of connective tissue, composed (mainly of collagen, a bar that may also include, depending on the tissue, other structural proteins, polysaccharides and mineral deposition: Isolation of cells from a given tissue necessarily requires the use of a technique for releasing cells from the connective tissue matrix. The isolation and purification of a specific type of differentiated cell therefore comprises two major stages; separation of cells from connective tissue matrix and separation of cells of the desired type from all other .20 types of cells found in tissue. The operating principles included and disclosed in the present invention will be applicable to the isolation of a variety of cell types from a variety of tissue sources. As an example, isolation of the Langerhans islands from the pancreas, suitable for isolating the mRNA code for insulin, will be mentioned.

Insulin-producing cells may also come from other sources, such as the calf-fetus pancreas or cells grown from tumors of the insulin. Isolation of pure cells ... it is simple in these cases, especially when it is. they use pure cell cultures. The method of. isolation of the cells from the islands, discussed above, would not be necessary in these cases; however, the method remains advantageous due to its general applicability.

Frequently, it will be found that the desired mRNA ratio can be increased by using the advantage of cellular response to environmental stimuli. For example, treatment with a hormone may result in increased production of desired mRNA. Other techniques include raising at a certain temperature and exposure to a specific nutrient or other chemical, in isolating growth hormone mRNA from rats, treating cells, cultured pituitary rats with thyroid and glucocorticoid hormone raised, synergistically. , the proportion of growth hormone mRNA with a significant amount.

2. MRNA extraction. An important feature of the present invention is the essentially complete removal of the activity of the RNase in the cell extract. The mRNA to be extracted is a unique polynucleotide chain, not paired with any complementary chain. Therefore, the bidrolytic cleavage of a single phosphodiester linkage in the sequence would render the entire molecule useless for the purpose of transferring an intact genetic sequence to a microorganism. As will be shown, RNase enzyme is widely distributed, active and exceptionally stable. It is found on the skin, survives the usual glass washing techniques and sometimes contaminates the deposits of organic chemicals. The difficulties are particularly acute when handling extracts of cells from the pancreas, as the pancreas is a source of digestive enzymes and is therefore rich in RNase. however, the problem of contamination with RNase is present for all tissues and the method described in this invention for eliminating the activity of RNase is applicable to all tissues. The exceptional efficacy of the method is demonstrated in the present invention by successfully isolating an intact mRNA from cells isolated from pancreatic islets.

The present invention uses in combination a chaotropic anion, a chaotropic cation and a disulfide bond breaking agent, during cell breakdown and during all the operations required for RNA separation, essentially protein free. The efficacy of the combined action of the aforementioned agents has been demonstrated by their use in isolating essentially non-degraded mRNA with good yields from Langerhans islands, isolated from rat pancreas.

The choice of chaotropic ions is based on their solubility in aqueous media and their availability. Suitable chaotropic cations include guanidinium, carbamoylguanidinium, guanilguanidinium, lithium and the like. Suitable chaotropic anions include iodide, perchlorate, thiocyanate, diiodsalicylate and the like. The relative effectiveness of the salts formed by combining such cations and anions will be determined, in part, by their solubility. For example, lithium diosalicylate is a stronger denaturant than guanidinium thiocyanate, but it has a solubility of only about 0 and M and is also relatively expensive. Guanidinium thiocyanate provides the preferred cationanion combination, because it is easy to obtain and is highly soluble in aqueous media, up to about 5 M.

Thiol compounds, such as 0rifl-mercaptoethanol, are known to be able to break the intramolecular disulfide bonds in proteins through a thiol-disulfide exchange reaction. Many suitable thiol compounds, known as , propanol-dimercaptan and the like. Solubility in water is a necessary property, as thiol must be in excess of intramolecular disulfides to essentially direct the exchange reaction to fulfillment, Rc / a-mercaptoethanol is preferred, due to the possibility of easy procurement at reasonable prices. .

In order to inhibit RNase during RNA extraction from tissue cells, the efficacy of a given chaotropic salt is directly related to its concentration. The preferred concentration is therefore the highest concentration that can be used, under practical conditions. The success of the present invention in the intact conservation of mRNA during extraction is considered to depend on the speed with which the RNase is denatured, along with the extent of the denaturation. The effectiveness of a denaturant is defined as the threshold concentration (critical), necessary to ensure the complete compression of a protein. On the other hand, the speed of denaturation of many proteins depends on the concentration of the denaturant, relative to the threshold, raised from the 5th power to the 10th power. Qualitatively, this relationship suggests that a denaturant only slightly stronger than guanidinium hydrochloride can distort a protein many times faster at the same concentration. The relationship between the kinetics of RNase distortion and the conservation of mRNA during its extraction from, cells is considered to have not yet been recognized or exploited prior to the presence of this gene. The preceding analysis, if coated, suggests that the preferred denaturant will be that with a low concentration (critical) denaturation concentration, combined with a solubility in. high water. For this reason, the guanidinium thiocyanate is preferred over the lithium diodsalicylate, although the latter is a stronger denaturant, due to the solubility of the guanidine thiocyanate, which makes it usable at a concentration that allows inactivation of the RNase. faster. The previous analysis further explains why guanidinium thiocyanate is preferred over soluble hydrochloride, the first being a somewhat stronger denaturant.

The use of a disulfide bond breaking agent in combination with a denaturant enhances and enhances the effectiveness of the second, allowing the RNase molecule to fully extend. The thiolic compound is considered to enhance the rate of advancement of the denaturation process, by preventing the rapid denaturation that might occur when the intermolecular disulfide bonds are left intact. In addition, any contaminated RNase, remaining in the mRNA preparation, will remain substantially inactive, even in the absence of denaturant and thiol. Disulfide bonding agents having thiol groups will, to some extent, be effective at any concentration, although, in general, a large excess of thiol groups is preferred over intramolecular disulphide bonds to direct the reaction of change in the direction of cleavage of intramolecular disulfides. On the other hand, many thiol compounds are bad smelling and it is unpleasant to work with them at high concentrations, so that there is practically a limit, higher concentration, When using β-mercaptoethanol, concentrations have been found to be effective. 0.05 M and 1.0 il and the concentration of 0.2 M is considered optimal for isolating undegraded RNA from rat pancreas. The H value of the medium during mRNA extraction from cells can be anywhere in the range H = 5.0 ... 8.0. Following the step of ru78200 pears of the cell, 'RNA is separated from the mass of cellular protein and DNA. For this purpose, numerous processes are known, any of which are suitable. A common method of treatment in the prior art is to use an ethanol precipitation method, which selectively precipitates RNA. The preferred technique of the present invention is to bypass the precipitation step and stratify the homogenate directly over a 5.7 M cesium chloride solution, in a centrifuge tube, then subject the tube to centrifugation (Glisin V. et al. Biochvmistry 13, 1974, p. 2 633). This method is preferred because a hostile RNase environment is maintained continuously and high-yield RNA is obtained, free of DNA and protein.

The working methods described above result in the purification of total RNA from the cell homogenate. but only a portion of this RNA is AKNm desired. In order to further purify the desired mRNA, it takes advantage of the advantage offered by the fact that, in the cells of the higher organisms, the mRNA, after transcription, is processed further into the cell by attaching a polyadenyl acid. Also, mRNA containing poly sequences attached to it can be selectively isolated, by cellulose column chromatography, which is oligotimidylated (Aviv H, Leder P. Proc .. Natl. Acad. Sei, USA, 69, 1972, p. 1 408). The above working methods are sufficient to provide essentially pure, intact and translatable mRNA from sources rich in RNase. Purification of mRNA and subsequent in vitro operations can be performed in essentially the same way with any mRNA.

In certain circumstances, for example, when cells from tissue cultures are used as mRNA sources, contamination with RNase may be quite low, so that the inhibition method of the described RNase is not required. In such cases, state-of-the-art methods for reducing RNase activity may be sufficient.

3. CDNA formation. For the schematic representation of the steps that remain to be executed in the process, reference is made to fig. 1. The first step in this process is the formation of a complementary DNA sequence on purified mRNA. The enzyme of choice for this reaction is reverse transcriptase, although, in principle, any enzyme capable of forming a complementary DNA strand using mRNA as a template could be used. The reaction can be carried out under the conditions. described in the prior art: - using mRNA as a template and a mixture of four deoxynucleoside triphosphates as precursors of the DNA chain24. It is advantageous to provide that one of the deoxynucleoside triphosphates is marked with 32 P in the alpha position, in order to be able to monitor the reaction rate, to provide a sign (mark) for the recovery of the product after the separation operations, such as chromatography and electrophoresis, and for the purpose of making a quantitative estimate of recovery (Efstradiadis et al., Cell 7, 1976, p, 279).

As illustrated in the figure, the reverse transcriptase reaction product is known ui â vi). double chain, in the form of a head needle with non-covalent link between the RNA chain and the DNA strand.

The reverse transcriptase reaction product is removed from the reaction mixture by standard methods known in the art. It was found useful to use a combination of extraction with phenol, chromatography on Sephadex G-100 and precipitation with ethanol.

Once the cDNA enzyme has been synthesized, the RNA template can be removed. Various processes for selective RNA degradation in the presence of DNA are known in the prior art. The preferred method is alkaline hydrolysis, which is highly selective and can be easily monitored by adjusting the gH.

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

The synthesis of a double stranded cDNA in the form of a head needle is performed by using a suitable enzyme, for example DNA polymerase or reverse transcriptase. The reaction conditions are similar to those described above, including: the use of alpha32 P-labeled nucleoside triphosphate, reverse transcriptase can be obtained from a variety of sources. A convenient source is bird myeloblastosis virus, which can be obtained through the National Institute for Health, USA.

After the cDNA head needle is formed, it may be advantageous to purify DNA from the reaction mixture. As described above, it has been found that the convenient method is to use phenol extraction steps, Sephadex G-100 chromatography and ethanol precipitation to purify the DNA product released from the contaminating protein.

The head-to-head structure can be transformed into an ordinary double-stranded DNA structure by removing the single-stranded loop, bringing together the ends of complementary strands. To this end, there are a variety of enzymes capable of performing specific hydrolytic cleavage of single-stranded DNA regions. A suitable enzyme for this purpose is nuclease Sf, isolated from

Aspergillns oryzae, Treatment of DNA, having the head needle structure, with A Sl nuclease, results in a high yield of cDNA molecules with paired base ends. Thereafter, extraction, chromatography and precipitation processes with alcohol are carried out, as described above. The use of reverse transcriptase and Si nuclease in transcriptchlor synthesis after double-stranded cDNA mRNA has been described in the literature (Efstradiadis A and CM 7, 1976, p. 279).

Optionally, the proportion of cDNA molecules having truncated ends can be increased by treatment with F. coli DNA polymerase I in the presence of the four deoxynucleoside triphosphates. The combination of the exonuclease enzyme and polymerase activities has the effect of removing any prominent 3 'ends and filling any 5' prominent ends. By this, the maximum proportion of cDNA molecules is ensured in subsequent ligation reactions.

The next step in the process involves treating the ends of the cDNA product to obtain sequences close to each end containing a recognition site for endonuclease dc restriction. The choice of the DNA fragment to be added at the ends is determined by the convenient manipulation modes. The sequence cc must be added at the ends to be chosen based on the endonuclear enzyme of special restriction and this choice depends, in turn, on the choice of the DNA vector with which the cDNA must be recombined. The chosen plasmid must have at least one position susceptible to restriction endonuclease cleavage. For example plasmid pMB-9 contains a restriction position for the Hind III enzyme. This Hind enzyme. III is isolated from Heinophilus influenzac- and purified by known methods (Wilcox et al. J. Mol. Biol, 51, 1970, p. 379 and Middleton JH]. Virol. 10, 1972, p. 42). Hac III enzyme is isolated from aigyptious Hemophiluy and is also purified by known methods (Wilcox'si others - J. Mol. Biol. 51, 1970, p. 379 and Middleton JHJ Virol. 10, 1972, p. 42) . An enzyme from Hemophilns snis, called Hsu I, catalyzes the same hydrolysis with a specific position, at the same recognition site as Hind. III. The two enzymes are therefore considered functionally interchangeable.

For the purpose of attachment, at the ends of the duplex, the cDNA is advantageous to use a chemically synthesized double-stranded decanucleotide, which contains the recognition sequence for Hind 'III Double-stranded Decanucleotide has the' sequence '¹ shown in FIG. 1. Specialists may find a variety of such synthetic sequences of restriction positions, so it is possible to prepare the ends of a DNA duplex so as to be sensitive to the action of a restriction endonuclease of such a wide variety.

Attaching the restriction position sequences to the cDNA ends can be carried out by any step known to those skilled in the art. The preferred method is the reaction called the truncated end ligand, catalyzed by⁴DNA ligase purified by a known method (Panel A et al. Biochemistry 12, 1973, p. 545). The ligation reaction of the truncated ends has been described in the literature. The product of the dc ligand reaction of the truncated ends between a truncated end cDNA and a double-stranded decanucleotide molar excess, containing the restriction position of the Hind III endonuclease, is a cDNA plating sequences of Hind III restriction positions at each end. Treatment of the reaction product with the Hind endonuclease arc results in a cleavage to the restriction position, with the formation of 5 'self-complementing single-stranded ends, as shown in Fig. 1,

4, The formation of a recombinant AD1 / dc transfer vector, in principle, a large variety of viral and plasmid DNAs could be used to form recombinations with a cDNA prepared in the manner described above. The main conditions are for the DNA transfer vector to be able to enter a host cell, subject to replication in the host cell and, in addition, to have a genetic determinant, by which it is possible to select those cell host that received the vector. however) for reasons of public health, the field of choice should be restricted to those species of transfer vectors that seem appropriate for the type of experience used, in accordance with the guidelines of the National Institute for Health, USA (Federal Register vol. 41, no. 131, July 7, 1967, pp. 27 902 - 27 943). The list of DNA transfer vectors is continuously expanding as new vectors approved by the relevant committee develop and it should be understood that the present invention considers'.the use of any viral DNA and plasmid that would have the capabilities described, including those to which the committee it will give them approval later. Suitable transfer agents, which are currently approved for use in the inode, comprise a variety of dc derivatives of the bacteriophage lambda (Glisin V. C et al. -'Biochemistry 13, 19/4, p. 2 633 The plasmids derived from. characterized as being relatively small, having molecular weight as the order of each mi78200 lions and its property the number of copies of the plasmid DNA per cell-host can be increased to 20 ... 40 under normal conditions to 1000 or more, by treating host cells with chloramphenieol. The ability to amplify the dosage of the gene in the host cell makes it possible, under appropriate circumstances, under the control of the researcher, to determine the host cell to produce mainly proteins encoded by the genes contained in the piasmide. Therefore, the derivatives of the collar El are preferred transfer vectors in the process according to the present invention. Suitable derivatives of the collar It comprises plasmids pMB-9, which carry the tetracycline resistance gene and pBR-313, pBR-315, pBR-316, pBR-317 and pBR-322 which contain, besides the tetracycline resistance gene, gene for resistance to ampicillin. The presence of some drug resistance genes provides a convenient way to select cells that have been successfully infected by the piasmide, because colonies of such cells can grow in the presence of the drug, when cells that have not received the plasmid they will not grow or form colonies. In the experiments of this invention, a piasmide derived (originating) from the neck of El was used, which contained, besides the marker described for drug resistance, a Hind III position.

As with the choice of plasmid, the possibility of choosing a suitable host is, in principle, very high, but very limited due to public security. A strain of E. coli designated with X-1776 was developed and received the approval of the National Institute for Health, USA, for the procedures of the type described in this invention, using P2 container facilities (Curtis-III R Ann. Rev. Microbiol 30, 1976, p. 507). E. RR-1 sheets are suitable when P3 container installations are available. As in the case of plasmids, it should be understood that the present invention considers the use of any host cell strains, having the ability to activate as a transferor for the chosen vector, including protists, other than bacteria, for example yeasts, whenever will be approved for use, according to the guidelines of the National Institute for Health, USA '

Plasmids for recombination (recombinant plasmid) are formed by mixing a restriction endonuclease-treated plasmid-DNA with a cDNA containing terminally treated groups; like, "To minimize the chance that the cDNA segments will form one-to-one combinations." the other, sc adds the plasmid DNA to one. molar excess of cDNA.

2S in the prior art, this resulted in most plasmids circulating without the inserted cDNA fragment. Subsequently transformed cells mainly contained the plasmid and not the cDNA for recombination. As a result, the selection process was very tedious and time consuming. The solution in the prior art to this problem was the attempt to design DNA vectors having a dc endonuclease position 'in the middle / of a suitable marker gene,' '- •• so that the insertion of a recombinant splits the gene, causing, by this, loss of gene-encoded function.

It is preferable to use a method of reducing the number of colonies to be protected for recombinant plasmids. The method comprises treating the DNA piasmide fraction with restriction endonuclease with phosphatase-alkaline, an enzyme that can be commercially available. Treatment with alkaline phosphatase removes 5 'terminal phosphates from the endonuclease-generated ends of the plasmid and thus causes DNA-plasmid self-ligation. Consequently, the formation of the circle, that is, the transformation, will depend on the insertion of a DNA fragment containing the 5 'phosphorylated ends. The described process reduces the relative frequency of the transformation in the absence of recombination to less than 1 compared to KU⁴ .

The present invention is based on the fact that the DNA-ligase catalyzed reaction takes place between a 5 '- phosphatic DNA terminal group and a 3' hydroxyl DNA terminal group. If the phosphatic 5-terminal group is removed, the binding reaction does not occur. When double-stranded DNA is to be assembled, three situations are possible, as shown in Table 1.

In Table 1, double-stranded DNA is schematically represented by the solid parallel lines, while the respective 5 'and 3' end groups are labeled as hydroxyl (OH) or phosphate (OPO).₃H₂), as the case may be. In case I, phosphates-5 'are at both ends of the reactant, so that both chains are covalently bonded. In case II, only one of the chains to be assembled possesses a phosphate-5 ', the result - being that a single covalent bond with a single chain is produced, leaving, on the other chain, a discontinuity break with a single chain. The covalently unbound chain remains associated with the reunited molecule, by virtue of hydrogen bond interactions between complementary base pairs on opposite chains, which is well known in this field. In case III, none of the ends, the reactant has no fos78200 fat-5 'and no binding reaction can take place.

By this, unwanted binding reactions can be avoided by treating the close ends of the annealing, the binding of which must be prevented, in order to remove phosphate-5 'groups from them. Any suitable method for removing the phosphate-5 'groups may be used, unless this otherwise damages the DNA structure, which must be used. Alkaline phosphatase enzyme catalyzed hydrolysis is preferred.

Table 1

<td>case</td><td colspan="2">reactants</td><td>The product of ligase</td>

<td>I</td><td> 3’</td><td> 5’</td><td> 3 * .5^r</td>

<td></td><td>...OH</td><td>Wo ...</td><td>O-p ... a ... + 2H, O</td>

<td></td><td>... ΟΡΟ., Η,</td><td>HO ...</td><td>... O — P— O ...</td>

<td></td><td> 5'</td><td> 3’</td><td> 5’ 3’</td>

<td>countries</td><td> 3'</td><td> 5'</td><td> 3' 5’</td>

<td></td><td>... 014 -r</td><td>A...</td><td>... O-P-O ... 3- H, O</td>

<td></td><td>...OH</td><td>OH...</td><td>... OH HO ...</td>

<td></td><td colspan="2"> 5¹ A '</td><td> .5' 3'</td>

<td>HI</td><td colspan="2"> 3’ 5’</td><td></td>

<td></td><td>...OH ... sheep 5'</td><td>HO ... -b HO ... 1 t A</td><td>the reaction does not take place</td>

The process described above is also useful in the case where a linear DNA molecule has to be cleaved into the fragments, typically, using a restriction endonuclease enzyme 1, then reconstituted in the original sequence. The subfragments can be purified separately and the desired sequence can be reconstituted by subgroup reunification. For this purpose, the DNA-ligase enzyme, which catalyzes end-to-end binding of DNA fragment, can be used.

Where the binding sequences do not have truncated ends, ligase obtained from E. coli (Federal Regisler vol. ²⁰41, no. 131 July 7, 1967, pp. 27 902 ....

... 27 943 and Modrich Y, Lehman IR,

J. Biol. Chem. 245, 1970, p. 3 626).

Efficiency of reconstructing the original sequence from subfragments produced by tra- ²⁵the restriction endonuclease region will be greatly increased by using a method of preventing the reconstruction in improper sequence. This undesirable result is avoided by treating the cDNA fragment^3Jthe desired homogeneous length and sequence with an agent capable of removing the terminal 5 'phosphate groups from the cDNA, before the homogeneous cDNA cleavage with restriction endonuclease. A phosphatase enzyme is preferred³⁵alkaline. The phosphate-5 'terminal groups are a precondition of structure for the subsequent DNA-Iigase binding action, used to reconstruct the cleaved subfragments. Therefore, the ends * with the lack of phosphate -5 'terminal cannot be covalently assembled. The DNA subfragments can be 'unified only at the ends containing phosphate-5' generated by the restriction endonuclease cleavage performed on the isolated DNA fragments.

The above process avoids the formation of the most significant, unwanted binding reactions, namely the union 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 avoided, as they can occur through a type II reaction, Table 1 above. Such secondary reactions are less annoying, because they lead to identifiable and physically separable products, when the recombination in the reverse order does not.

In order to illustrate the ways described above, a cDNA for rat insulin was isolated and recombined with a plasmid. DNA molecules were used to transform E. coli X-1776. The transformants were selected by growth on a tetracycline-containing medium. A recombinant DNA plasmid obtained from transformed cells was found to contain an inserted DNA fragment of approximately 410 nucleotides in length. Other recombinants were obtained, isolated by the same way of working and analyzed. The inserted fragments were released from the plasmid by digestion in endonucleases Iiind IIΓ or IIsu 1 and subjected to DNA sequence analysis by a known method (Alaxani AM, Giibert W. Rroc. Natl. Acad. Sci USA, 74, 1977, p. 560), it was found that the nucleotide sequences of the inserted DNA fragments overlap and that they contain the entire coding region of rat pro-insulin I, as well as 13 of the 23 amino acids of the prepeptide sequence. An assembly of the nucleotide sequence in this region has been constructed, which is shown. The theoretical bases of the invention are as follows.

The biological significance of the DNA base sequence is that it represents a repository of genetic information. The DNA base sequence is used as a code that specifies the amino acid sequence for all proteins made by the cell, in addition, portions of the sequence are used for regulatory purposes, to control the coordination in time and the amount of each protein formed. The understanding of these control elements is only partly understood. In conclusion, the base sequence of each strand is used as a template for DNA replication that accompanies cell division.

The way in which DNA base sequence information is used to determine the amino acid sequence of proteins is a fundamental process that, in its broad lines, 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 DNA segment containing a triplet sequence corresponding to the amino acid sequence in the protein. The genetic code is shown in Table 2 below.

In the biological process of transforming information from the nucleotide sequence into the structure of the amino acid sequence, a first step, called transcription, is executed. In this step, a local DNA segment, having a sequence specifying the protein to be made, is first copied with RNA. RNA is a polynucleotide similar to DNA, except that deoxyribose is replaced by ribose, and instead of thymine, uracil is used. RNA bases are able to enter in the same way as κD pairing bases that exist in DNA. Accordingly, the RNA transcript of a DNA nucleotide sequence will be complementary to the copied sequence. Such an RNA is called messenger RNA (mRNA), because of its intermediate location between the genetic apparatus and the protein synthesis apparatus in the cell.

Inside the cell, mRNA is used as a template in a complex process involving a multiplicity of enzymes and organs within the cell and resulting in the synthesis of the specific amino acid sequence. This process is called mRNA translation,

Often there are additional steps, called processing, which are executed for transforming the amino acid sequence synthesized by the translation process into a functional protein. In the case of insulin, the process of synthesis in the body follows:

Genetically

<td>Phenylalanine (Phe)</td><td>TTK</td><td>His (His) history</td><td>CAK</td>

<td>Leuctna (Leu)</td><td>χία;</td><td>Glntnjnina (Glu)</td><td>CAJ</td>

<td>Leucine Island (s)</td><td>ATM</td><td>AspaAgină (Asii)</td><td>ΛΑΚ</td>

<td>Meliouin (Met)</td><td>ATG</td><td>Factory (Lys)</td><td>AAJ</td>

<td>Y alina- (Wave)</td><td>GTL</td><td>Aspartic acid (As.p)</td><td>GAK</td>

<td>Serin</td><td>OK</td><td>Glutamic acid (Gb)</td><td>CAJ</td>

<td>Prdinâ (Pn)</td><td>CCL</td><td>Cystine (Cys)</td><td>TGK</td>

<td>Threonine (Th.i</td><td>ACL</td><td>Tripiophan (Tryl</td><td>IGG</td>

<td>(9)</td><td>GCL</td><td>Αιτζΐηίιι &amp; (Light</td><td>WGZ</td>

<td>Tyrose (Tyr)</td><td>TA that</td><td>Glycine Î / lly)</td><td>GCL</td>

<td>Termination signal</td><td>Taj</td><td></td><td></td>

<td>End signal</td><td>TGA</td><td></td><td></td>

The key: Each 3-letter triplet represents a trinucleotide of DNA »having a 5 * left end and a 3 'end on the right. The letters represent the purine or pyrimidine bases that form the nucleotide sequence. A - adenine; G = guanine; C = cytosine; T - limlnâ; X - T or C if Y is A or G; X - c if Y is C or Τ; 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,

W is a; QU TC if A, G, C or T;

G. C or T if QT is TC; s - T or C or C ,; L - A, T, C or G; lvî A, C or T.

The immediate precursor of insulin is a single polypeptide, called proinsulin, which contains the two chains of insulin A and B, which are joined by another peptide.

C or T if W assigns C, Z = Λ or G if QR = AG if S is T or C; a = A if OR assign AG; J = A or G; K = T (Steiner DF, Cunningham D, Spigelman L, Atheus B, Science 157, 1967, p697). It has recently become known that the initial translation product of mRNA insulin

782CO is not proinsulin itself, but a pro-insulin that contains more than 20 additional amino acids on the amino terminus of proinsulin (Calin S. J ,, Keim P, Steiner D. F, Proc. Natl. Acad. Sci USA 73, 1976, ρ I 964; Lomedico Ρ. T., Saunders GF, Nuci. Adds Res. 3, 1976, p. 381). The structure of the preproinsulin molecule can be schematically represented as follows:

NH₂ - (pre-peptide) - chain B - (peptide C) - chain A - COOH.

Many proteins of medical importance or importance for research are found in or are made by the cells of higher organisms, as is the case with insulin, including enzyme-catalyzed reactions. The nature of these enzymatic reactions, as they are understood in the prior art, is described below.

Reverse transcriptase catalyzes the synthesis of complementary DNA against an RNA template, in the presence of the RNA template, an olido-deoxynucleotide primer and the four deoxynucleoside triphosphates, dATR, dGTP, dCTP and TTP. The reaction is initiated by the noncovalent linkage of the olido-deoxynucleotide primer at the 3 'end of the mRNA followed by the stepwise addition of the appropriate deoxynucleotides, as determined by the base pairing relationships with the nucleotide sequence of the 3' mRNA. The produced molecule can be described as a head needle structure, containing the original RNA together with a complementary strand of DNA linked to it by a single strand 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 single stranded DNA loop. head set (Aviv H, Suder P., Proc. Natl. Acad. Sci, li.SA 69, 1972, p. 1408) Efstradiadis A., Kafatos FC, Maxam A. lb, Maniatis T, Ccll. 7, 1976, p. 279).

Restriction endonucleases are enzymes capable of hydrolyzing phosphodiester bonds in double stranded DNA, thereby creating a continuity interruption in the DNA chain. If the DNA is in the form of a closed loop, the loop is transformed into a linear structure. The main feature of such an enzyme is that its hydrolytic action is applied only at a point where a specific nucleotide sequence is encountered. Such a sequence is called the recognition position for restriction endonucleases. Endonucleases were isolated from restriction from a variety of sources and they were. characterized. according to the nucleotide sequence of their recognition positions.

restriction endonuclear lines hydrolyze phosphodiester bonds on both chains at the same point, producing truncated ends. Others catalyze hydrolysis to separate bonds from each other by several nucleotides, producing single chain free regions at each end of the cleaved molecule. Such single-stranded ends are self-complementary, so cohesive, and can be used to reunite with hydrolyzed DNA. Since any DNA susceptible to cleavage by such an enzyme skilled in the same recognition position will produce the same cohesive ends, so it will be possible to bind heterologous DNA sequences, which have been treated with endonuclease. restriction, with other sequences treated similarly. (Robcrts RJ Crit. Rev. Biochern. 4, 1976 p. 123). Restrictive positions are relatively rare; however, the general utility of restriction endonucleases has been greatly amplified by the chemical synthesis of double-stranded oligonucleotides, which present the sequence with the restriction position. Therefore, effectively any DNA segment can simply be coupled to any other segment by simply attaching the appropriate restriction cligonucleotides to the ends of the molecule and subjecting the product to the hydrolytic action of the appropriate restriction endonuclease, thus producing the required cohesive ends (Heyneker HL saNature , 263, 1976, p. 74S; Scheller Ρ, H. et al., Science 196, 1977, p. 177; Vogt V. m '., Eur. /. Biochem, 33, 1973, p. 192)

Endonuclease S1 is an enzyme of general specificity, capable of hydrolyzing phosphodiester linkages from single-stranded DNA or from loops or single-stranded loops of otherwise double-stranded DNA (Federal Registered Vol. 41, No. 131, July 7, J967, pp. 27 902 ... 27 943).

DNA ligase is an enzyme capable of catalyzing the formation of a phosphodiester bond between two DNA segments, yielding a 5 'phosphate and a 3' hydroxyl respectively, so that two DNA fragments held together by cohesive ends can be formed. The normal function of the enzyme is thought to be the binding of single-stranded strands in a DNA molecule, otherwise in the double chain. However, under appropriate conditions, DNA ligase is capable of catalyzing the truncated end bond in which two truncated end molecules are covalently bonded,

Alkaline phosphatase is an enzyme with general specificity capable of hydrolyzing phosphatic esters including 5 'terminal phosphates of DNA.

A step further in the general process to be described is the insertion of a specific DNA fragment into a

782C0 DNA vector, such as a plasmid. Plasmid is the term given to any unit of DNA that replicates autonomously and can be found in a microbial cell, other than the host cell genome itself: A plasmid is not genetically linked to the host cell chromosome. Plasmid DNA exists as double-stranded ring (ring) molecules, generally of the molecular weight order of several million, although some are larger than the IO® molecular weight,_eand_e usually represents only a small percentage of the total DNA of the cell. The plasmid DNA can be separated from the DNA of the host cell, due to a large size difference between them.

Plasmids can be replicated independently of the host cell division rate and, in some cases, their rate of multiplication can be controlled by (controlled by) the researcher, through variations in growth conditions. Although the plasmid exists in the form of a closed ring, it is possible to insert, by artificial means, a DNA segment into the plasmid, forming a recombinant plasmid as an enlarged molecular size, without substantially affecting its ability to bind. replication or to remove any genes he could wear. Therefore, the plasmid serves as a useful vector for transferring a DNA segment to a new host cell. Plasmids that are useful for DNA recombination technology typically contain genes that may be useful for selection purposes, such as genes for drug resistance.

however, until the present invention there was no technique for introducing the insulin gene into a bacterium. 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, so that the specific protein finally produced is also modified. This one. it would be possible to produce, for example, an insulin tailored to, to suit a specific medical need, For this, the genetic ability to produce any sequence of insulin-related amphnoacids, having the essential functional properties of insulin, may be conferred on a microorganism .

The symbols and abbreviations used in the present description are presented below: DNA - deoxyribonucleic acid;

RNA - ribonucleic acid;

CDNA - complementary DNA (synthesized enzymatically from seventy mRNAs):;

A - adenine;

T - thymine;

G - guanine;

ArNm - messenger RNA;

TRNA - transfer RNA; daTP - deoxyadenosine triphosphate; dGTP - deoxyguanosine triphosphate; dCTP - deoxycytidma triphosphate;

A - adenine;

T - thymine;

G - guanine;

C - cytokinase · Tris ·· - 2-amino-. Hydroxy-ethyl-1,3-propane diol; '· /

BDTA - ethylenediamine-tetraacetic acid; ATP - anosine triphosphate;

TTP - thymidine triphosphate dc;

Alu I position - A.GjCT sequence, specifically cleaved at the arrow through the Aon endonuclease;

Ham HI position - GjGATCG sequence, specifically cleaved at the arrow by endonucleases Bam HI;

Position Bgl Ϊ - the GCCNNNNf NGGC sequence, specifically sequenced to the arrow by Bgl · I endonuclease (Note: N refers to any nucleotide);

Eco RI position - GfAATTC sequence, specifically cleaved at the arrow by R'I endonuclease;

Eco RII position - ICCAGG or | CGTGG sequence specifically cleaved at the arrow by Eco RII endonuclease;

Position Hac II - the sequence AGGGC + T 'or AGCGCțCsau GGCGGjT or

GGCGGIC, specifically cleaved to the arrow by Hae II endonuclease;

Hinc II position - GTTAAC or GTTGAc or GTCAAG or GTCGAC sequence, specifically cleaved by Hinc II endonucleases;

Pst position I - the sequence CTGCA1G, specifically cleaved at the arrow by endonucleases Pst I;

Position I Sal - GjTCGAC sequence, specifically cleaved at the arrow by endonucleases Sal

Position Ssf I - GAGCTjC sequence, specifically cleaved at the arrow by endonucleases Sst I.

The invention has the following advantages:

- gives a microorganism the genetic ability to produce any amino acid sequences related to · insulin, having essential functional properties of insulin;

- creates the possibility of establishing - symbiotic relationships between the microorganisms produced according to the invention and human beings with chronic or - acute deficient diseases, the microorganisms modified - genetically obtained thus being implanted in - or otherwise associated with - a person in order to: compensate pathological deficiency in its metabolism;

- creates the possibility of obtaining unlimited quantities of insulin, because microorganisms such as bacteria are relatively easy to grow in chemically defined environments;

- fermentation technology is very advanced and can be well controlled;

- the growth of organisms is rapid and high yields are possible; in addition, certain microorganisms have been very well characterized genetically and are, in fact, among the best characterized and most well understood organisms; In addition, the possibility to isolate and transfer the genetic sequence that determines the production of a certain protein to a microorganism, which has a well-defined genetic background, provides a valuable research tool for studying how the synthesis of such a protein is controlled and how the protein is processed after synthesis; also, isolated gene sequences could be modified to encode for different proteins having modified therapeutic or functional properties.