Method of cdna fragment cleaning with specific nucleotide sequence

Proteins and peptides are synthesized almost in an infinity of varieties in living organisms. Many of these have proved to be of medical, industrial or agricultural utility. Some of the proteins are enzymes, used as specific catalysts for complex chemical reactions. Others function as hormones, which act on the growth or development of an organism or on the functioning of specific tissues in medically important ways. Proteins with specific links may be of commercial importance for the isolation and purification of trace substances and for the elimination of contamination substances. Both proteins and peptides are composed of linear amino acid chains, the latter term being used to shorten single-chain sequences, the first referring to multilayer or long-chain substances. ₂₅

Peptides and proteins are generally high molecular weight substances, each of which has a specific amino acid sequence. Except for smaller peptides, peptide and protein synthesis are often impractical, costly and time consuming, if not impossible. In most cases, in order to make practical use of the desired protein, it must be further isolated from the body that produces it. Frequently, the desired protein is presented only in small quantities. Often, the source organism cannot be obtained in sufficient quantities to achieve an adequate amount of desired protein, as a result, many of the potential medical, industrial and agricultural potential applications of specific proteins are known, but remain, in practice, unsolved, because there is no adequate source of protein and peptide.

Recently or, more recently, recently developed techniques have made possible the use of microorganisms, capable of rapid and abundant growth, for the synthesis of proteins and peptides78407

THE PRICE OF LEI 51.89 their lolosiloiire from the point of view of the corneas. These techniques make it possible to genetically equip a suitable microorganism with the ability to synthesize a protein or a peptide normally prepared by another organism. Technique uses a fundamental relationship that exists in all living organisms between genetic material, usually DNA and proteins synthesized by the body. This relationship is - in this way, so that the amino acid sequence of the protein is reflected in the nucleotide sequence of the DNA. There are one or several groups of trinucleotide sequences, specifically linked to each of the twenty amino acids, which occur most commonly in proteins. The specific relationship between each given trinucleotide sequence and its corresponding amino acid constitutes the genetic code. The genetic code is believed to be the same or similar for all living organisms. As a consequence, the amino acid sequence of each protein or peptide is reflected by the corresponding nucleotide sequence, according to the well-known relationship. Moreover, this nucleotide sequence can

be, 'in principle, transcribed by any living organism.

Table 1 gives the genetic code for a number of substances.

Table 1

<td>Substance</td><td>The genetic code.</td>

<td>Phenylalanine;</td><td>TTK</td>

<td>Leucine (Leo)</td><td>XTY</td>

<td>Isoleucine (Ile)</td><td>ATM</td>

<td>Methionine (Died)</td><td>ATG</td>

<td>Valina (Wave)</td><td>GTL</td>

<td>Serine (Serum)</td><td>QRS</td>

<td>Proline (Pro)</td><td>CCL</td>

<td>Treomin '(Thr)</td><td>ACL</td>

<td>Al, anina (Alin)</td><td>GCL</td>

<td>Termination signal</td><td>TAJ</td>

<td>Termination signal</td><td>TGA</td>

<td>Histidine (Hisj)</td><td>CAK</td>

<td>Glutamine (Glin)</td><td>CAJ</td>

<td>Asparagine (Asin)</td><td>AAK</td>

<td>Lysine (J, ys)</td><td>AAJ</td>

<td>Aspertic Acid (Asp)</td><td>GAK</td>

<td>Glutamic acid (Glu)</td><td>PLEDGE</td>

<td>Cysteine ​​(Cys)</td><td>TGK</td>

<td>Tryptophan (Try)</td><td>TGG</td>

<td>Arginine (Arg)</td><td>WGZ</td>

with a 5 'end on the left and a 3' end on the right. The corresponding letters represent the bases of purine or pyrimidine, which form the nucleotide sequence.

A = adenine

G = guanli | nă

C = cytosine

T = female

J = A or C; K = T saii C

L = A, T, C or G

M = A, C or T

X = T or C if Y is A or G

X = C if Y is C or T

Y = A, G, C or T if X is C

Y = A or G if X denotes T

W = G or A if Z is A or C

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 &lt;C

S = A, G, C or T if QR is TC 'S = T or C if QR is AG,

The Trinucleotides in Table 1, called codons, are presented as DNA trinucleotides, as they exist in the genetic material of the living organism. Expression of these codons in protein synthesis requires the intermediate formation of a • messenger RNA (mRNA). The mRNA codons have the same sequences as the DNA columns in table 1, except that the uracil is found instead of thymine. The complementary trinucleoitidine DNA sequences, having opposite polarity chains, are functionally equivalent to the codons in Table 1, as is known to those skilled in the art. An important and well-known aspect of the genetic code is its degeneration, whereby most of the amino acids used for the preparation of proteins can be used more than one for encoding the nucleotide triplet. As a result, a number of dc nucleotide sequences may be the code for a given amino acid sequence. Such nucleotide sequences are considered functionally equivalent, as they may result in the production of the same amino acid sequence in all organisms, although some chains may transcribe some 'sequences more efficiently than others would. Occasionally, a meitidate variant of purine or pyrimidine may be detected in a given nucleotide sequence. Such methylations do not in any way affect the coding relationship.

It is worth noting that the method of equipping a microorganism with the ability to synthesize a new protein is known in three general stages: (1) isolation

KEY: Each three-letter triplet represents a DNA trinuoleotide, and purification of the specific gene or nucleotide sequence containing the genetic information encoded for the amino acid sequence of the desired protein; (2) recombining the nucleotide sequence with a suitable transfer vector, typically the DNA of a bacteriophage or a plasmid, (3) transferring the vector to a suitable microorganism and selecting a receptor microorganism strain, which contains the desired genetic information.

A fundamental difficulty encountered in the attempts to commercially exploit the process described above: takes place in the first stage - isolation and purification of the desired specific genetic information. DNA exists in all living cells in the form of extremely high molecular weight nucleotide chains. A cell can contain more than 10,000 structural genes, encoding for amino acid sequences over 10,000 specific proteins, each gene having a sequence of several hundred nucleotides in length. For the most part, four different nucleotide bases gave rise to all the existing sequences. These are adienine (A), guanine (G), cytosine (C) and thymine (T). The long sequences comprising the structural genes of the specific proteins are consequently very similar in the entire chemical composition and physical properties. Separating such a sequence from the plethora of other sequences present in the isolated DNA can usually be accomplished by conventional physical and chemical preparation methods.

MRNA functions in the processes of converting nucleotide DNA information into the amino acid sequence structure of the protein

In the first stage of this process, called transcription, a local segment of DNA, which has a nucleotide sequence, specific to a protein, to be prepared, is first copied into RNA. RNA is a polynucleotide similar to DNA, except ribose is substituted with deoxyribose, and¹ uracil is used instead of thymine. The nucleotide bases in RNA are able to enter in the same way as the base pairs, according to / to the sons that are known to exist between complementary strands of DNA. A and U (T) are complementary, and G and C are also complementary. The RNA transcribed by a nucleotide DNA sequence will be complementary to the copied sequence. Thus, RNA is defined with messenger RNA, because of its status as an intermediary between the cell's genetic apparatus and its protein synthesis apparatus. Generally, only the mRNA sequence present in the cell at a given time is that which corresponds to the active protein synthesized at that time. Thus, a differentiated cell whose function is, first of all, to synthesize a single protein, will contain, first of all, the RNA species that correspond to that protein. Isolation and purification of the appropriate nucleotide sequence, which encodes a given protein, may be accomplished by isolating the mRNA and coding for specialized synthesis of such a protein in differentiated cells.

A major disadvantage of the procedure described above is that, it is only applicable in relatively rare cases, where cells can be found engaged in the primary synthesis of a single protein. Most of the proteins that are of commercial interest are not synthesized by their own means. The desired proteins may be one of a hundred or different proteins, which are produced by cells of a network or organism at a given time. Undoubtedly, the RNA isolation technique is potentially useful, since the establishment of the RNA species present in the cell is usually only a filler of the total sequences that exist in the DNA and thus performs an initial purification. In order to have the advantage of such a purification, a method is required whereby, even in cases with sequences present at a low frequency, so at a small percentage, they are isolated in a high purity.

for the purpose of genetic modification of microor^!ganisms) for such modified microorganisms to be used in practice, in literature, is cited Patents, France ur. 2281426; 2423541; RSR patent, no. 78200.

The present invention extends the range of processes aimed at obtaining human protein-producing microorganisms by performing a process of purifying a cDNA fragment having a specific deoxynucleotide sequence, which encodes human chorionic somatomamotropin, for recombination with nn vector DNA. transfer and transfer to a microorganism, comprising the steps of isolation of human placental tissue, isolating a cDNA population from this human placental tissue, synthesizing a cDNA population from this mRNA, and purifying cDNA, which encodes the human chorionic somaitomamoLion from the cDNA population, in which purified cDNA represents a minority of the population. of said cDNAs and at least a portion of these cDNAs has at least two restriction sites, wherein the cDNA population is heterogeneous in length and sequence and is prepared from said mRNA population, which is heterogeneous in length and sequence, using a reverse transcriptase catalyzed reaction, in which cDNA encoding human chorionic somatomamotropin is purified using: step a) of enzymatic hydrolysis of the cDNA population specifically at the restriction loci, by incubating a reaction mixture containing the cDNA population and a specific endonuclease for these restriction loci, namely by incubating the population mentioned with Hae HI at pH = 7 at 37 ° C for 1 to 2 hours to produce cDNA fragments; step b) fractionation of these cDNA fragments according to their length, namely, by gel electrophoresis, thus separating the cDNA fragments into fractions containing cDNA fragments of homogeneous length; step c) collecting the fraction containing the cDNA fragment encoding human chorionic somatomamotrophin, this fraction predominantly containing the respective fragment.

Four examples of embodiments of the invention are given in connection with FIG. 1 ... 5, which represents:

Fig. 1 shows the autoradiogram of a series of gel electrophoresis made with cDNA labeled with ³²p, as will be described in detail in Example 1;

FIG. 3 is a schematic representation of the nucleotide sequence encoding HCS, showing the relative locations of the different restrictive networks, as described in detail in Example · 1;

FIG. 3, an autodiogram of the results of gel electrophoresis, using cDNA labeled³²p, as will be described in detail in Example 2;

FIG. 4 and 5, autoradiograms of the results of the electrophoresis performed using cDNA labeled³²p, as will be described in detail in Example 3.

Example 1. In the text of the invention, the following abbreviations are used:

DNA = deoxyribonucleic acid

RNA = ribonucleic acid

CDNA = complementary DNA (enzymatically synthesized from a mRNA sequence)

MRNA = messenger RNA 'dATP = deoxyadenosine triphosphate dGTP = dioxiguanosine triphosphate dCTP = dioxycytidine triphosphate

HCS = human chorionic somatomamotropin

ATA = trichloroacetic acid

HGH = human growth hormone

A = adanine

T = thymine,

G = guamine C = cytosine U = uracil

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

EDTA = ethylenediaminetetraacetic acid,

ATP = adenosine triphosphate dTTP = thymidine triphosphate

The general procedure for isolating a specific cDNA sequence was demonstrated by isolating a sequence comprising a portion of the HCS coding region, extracted from placental tissue,

MRNA extracted from the placenta.

The human placentas obtained from the caesarean section were rapidly frozen in liquid nitrogen and kept at a temperature of -6B ° C. For extraction

Of the total RNA, 40 g of frozen placenta tissue was broken into a small piece and dissolved with a stirrer in 140 ml of 7 M guanidinium hydrochloric acid preparation (Cox RA, Metods in Enzymologv 12, 120 of 1908), 20 mM iris-HCl, pH 7, 5, 1 mM EDTA, 1% saciroisil (trademark Ci ba-Geigy Corp. Greensbore,

NC) a 0PC. After the addition of 0.5 g CaCl to each ml, the black-brown solution was heated at 65 ° C for 5 minutes, cooled rapidly in ice, transformed into a layer by the upper part of a^j5 ml of 5.7 CaCl, 10 mM irisHCl, pH = 7.5, 1 mM EDTA in a 2.54 χ 7.62 - 1.27 cm nitrocellulose tube and centrifuged in a SW 27 rotor ( Beckman Instruments Corp. Fullerton, California) at a speed of 27,000 rpm for 16 hours at a temperature of 15 ° C (Glisin, V. et al., Biochim., 13, 2633, 1974). After centrifugation in the tube, it was decanted, the tubes were drained, and the bottom part, containing 1/2 cm clear pellet RNA, was cut with a razor blade. The scales were transformed to a sterile Erlenmayer tube and dissolved in a 20 ml solution of 10 mM fris-HCl, pH = 7, 5, 1 mM EDTA, 5% sarcosyl and 5% phenol. Then the solution was poured into 0.1 M NaCl and stirred vigorously with a 40 ml mixture of 50% phenol - 50% chloroform. The RNA was precipitated from the anionic phase with ethanol, in the pyrazofence of 0.2 M Na-acetate pjH = 5.5. RNA strands were washed with 95% ethanol, dried and dissolved in sterile water. Usually, 40 g of placental tissue yielded about 30 mg of RNA, of which approximately 300 μ g of polyadenylated RNA were obtained after double chromatography on cellulose oligo-dT.

CDNA synthesis

The analytical reactions were carried out in 5 1 containing 50 mM fris-HCl, pH = 8.3; 0.1 mM EDTA; 7 mM MgCh; 20 mM KC1; 10 mM β-mercaptoethanol, · 40 μM dCTP (50 000 cpm³² p per mol); 500μ M for each dCTP, dATP and dTTP; 100 μ9 / ιη1 of polyadenylated RNA; 20 μ9 / πι1 incorporation dT₁₂._S and 100 units / ml of reverse transcriptase from avian myeloblastosis virus. The enzyme is available through the procedure given in Kacian DL and others in Methodes in Enzymology 29 and L. Grossman and others in Academic Press, New York, 1974 p. 15 C. Reactions were started by adding the enzyme at 0 ° C, and the syntheses were for 6 min at 42 ° C. under these conditions, about 10 ° cpm³²p were incorporated into the precipitable ATC material and each microgram of RNA resulted in 50 mg of cDNA. To obtain enough cDNA for sequence analysis, their reaction volumes were increased to 100 μΐ, and the dCTP concentration was increased to 250 μΜ (specific activity of 500 cpm).³²p per mol). Under these conditions, about 200,000 cpm dCMP labeled with cDNA were incorporated into the cDNA³²p.

Treatment with restriction endonuclease

For restriction endonuclease digestion, the analytical reactions were stopped by adding 20 μl of ice-water, boiled for 2 min, rapidly cooled to ice or, better, on ice, and passed 7 mM in MgCh . Aliquots (5 "1, about 2χ10⁵ cpm) were digested using an excess amount of endonucleases (endonucleases) Hae III or Hha I both for one hour at 37 ° C. Hae II was prepared according to the method given in Middleton JH et al. III, J. Vi'rol, 10, 42, 1972). The amount of enzyme used was determined empirically, to be in excess of the amount required for complete digestion of an equivalent amount of sensitive-restrictive DNA under identical reaction conditions. The reactions were stopped with 5 of 20µ 1 EDTA, 20¼ sucrose, 0.5¼ btromophenol blue, heated to 100 ° C for one minute and then analyzed by polyacrylamide gel electrophoresis. The products were separated on a weak gel compound

4.5 ... 10% polyacrylamide, for 2.5 h, at 150 V in fris-borate-EDTA buffer solution (Dingmam, CW, and Peakock, A "C., Biochemistry 14, 3787, 1957 -

Maniatis T., Jeffrey A.) and visualized by autoradiography of the dry gel.

Fig. 1 shows the results of gel electrophoresis and cDNA autoradiography³²p, prepared as described above. The samples were initially stained at the origin and migrated electrophoretically by 4.5¼ and then by 10% acrylamide. A bar is placed on the left side of the figure to indicate the position of the boundary between the two gel regions. Track A represents the electrophoretic migration of the total transcribed cDNA. Track B shows migration of cDNA treated with Hha I. Track C shows migration of cDNA treated with Hae III a. Track D shows the electrically oretic migration of total cDNA, treated with both Hha I and Hae III. Track E demonstrates electrophoretic migration of the isolated material from the prominent band of track C. Track F shows the electrophoretic migration of the material, isolated from the prominent band of track C, after treatment with Hha I. Track G shows electrophoretic migration of Hae III, cleaved c 5 ' -³²p marked M 13 single-stranded DNA, used for or as standard size, according to Hortiuchi, K. et al., Proc. Nat. Acad. Sci. USA, 72,555 since 1975. The approximate lengths of the nucleotides in these DNA fragments are indicated by the numbers on the right.

The results from lane A show that cDNA transcribed from placental tissue with mRNA is stereodispersed. Treatment with Hha I lane B or cU Hae III lane C results in the accumulation of polynucleotides of described lengths. Products of such discrete beads indicate the presence, in a heterogeneous population of transcribed cDNA, of at least one sequence present in multiple copies and which have two restriction networks for Hha I and Hae III respectively. Cleavage with Hha I produces a fragment of about 470 nucleotides, and digestion with Hae III produces a fragment of about 550 nucleotides in length. The digestion of both enzymes produces three fragments designated A, 90 nucleotides long B, 460 nucleotides long and C, about 10 nucleotides long. Due to its small size, the fragment C migrates from the gel under the conditions used in FIG. 1. The band of material that appears at the interface between 10% and 4.5% gel represents the heterogeneous material that was so large as to enter 10% gel and therefore accumulated at the interface. When the single band in track D is discussed, fragments A and B resemble the original one from the same cDNA molecule. This conclusion was confirmed by elu11 and a more fragment of 1.l gel I.ll from gel, which migrates as shown on track E, followed by rediges tia cil llha I. 'Uu such treatment produces two fragments. comigra all with strips (Jjbcrabilc through digestion '' combined .a total cDNA with Hâe þi and 1¾³ XU? How can the comparison of tracks D and F. be inclined? In total cDNA digestion, track D, the density of radiographs, which is a measure of the radigactiyitgta 'to! Qle present in the band, is higher for the fragment. It is only pegitru .fragment B, although the reverse has been expected on the table of different dimensions. This .pbşervațte suggests that the fragment  'is dranspreys from a darker regi .die end 3' of the mRNA than the fragment B.

Fig. 2 is a schematic representation or representation of the Aj> Nc molecule showing the relative location of the Hae III and Hha restriction networks 1. DNA fragments. A and B, derived from the same cDNA molecule, were ordered on d^flfor their intensity, they relate to the autoradiogram shown in fig. L track p. The existence of DNA fragment C, was interfered with by the difference in the electrophoretic mobility of the band that appears, in track 9 and track D in fig. 1. The dimension of the fragment A is known exactly from the determination of its nucleotide sequence by the method given <Ae Mg ^ am, A.pf. and Gilbert, W „Pioc. Nat. 1. Acad Șei USA ,, 74 560/1977. The size of fragment B, of DNA has been determined <in comparison with the size of the markings Ml 3 of DNA of_;roasted on fig · 1, track G.

The nucleotide sequences of fragment A and a portion of the 5 'end of fragment B were determined by the procedure given in Maxam, AM and Gilbert,' W., Proc; Nat. Acad. Sci. USA, 74 560/1977. Because the amino acid or HGS sequence is known, the nucleotide sequences of the two fragments will be compared to the amino acid sequence, using known relationships of the genetic code. Based on these relationships, it has been shown that the specific sequence gives, in fact, the code for the po: sions of the HCS molecule and confirmed by their ordering, fragments shown in FIG. 2.

Exemption 2 »The ability of the process, according to the present invention, to 'purify a desired huttoothiological sequence, which is a very small proportion of the nucyotide sequence in the proportion, is to be attained by the following reconstruction experiment: the mixture defined by NRA contains purified mouse globin RNA and pla11 RNA. In human polyadenylated RNA, they have been used as a template for reverse transeriptase, in the presence of ³²p dC TP. -.actiyițațe final apeciiien lb⁵ cpm per mol. The .de, cDNA products have been released with Hae III endonuclease, and. The cleavage products were separated on a slab gel composed of 4.5 ... 1Wo polyacrylamide. The cDNA fragments were visualized by autoradiography of the mixed gel.

Fig. 3 shows the results of these experiments. The pomegranates were prepared in ippd (essentially by the method (described in example 1. The dimensions of the markers prepared by cleavage with endonuclease. Hae III from phage Ml 3 DNA and 5'-).³²p, marked end, of the fragment formed by it, passed in lanes A and H. The approximate lengths of the nucleotides of these DNA fragments are indicated by; the numbers on the left-hand side. Tracks, 9 — G show the electric combs of rice produced through. initiation of the above sequence of the reaction with mixtures of globin RNA and placental RNA in varying proportions, as shown below:

<td>Track</td><td>Globin RNA (nanogram)</td><td>Plaenary RNA (nanograms)</td>

<td> ®</td><td> .300</td><td>of</td>

<td>. c</td><td> 60</td><td> 240.</td>

<td>D</td><td> 30</td><td> 270</td>

<td>E</td><td>¹⁵</td><td> 233</td>

<td>F</td><td> 7,5</td><td> 292,5</td>

<td>G</td><td> 0</td><td> 300</td>

It can be seen that the Hae UE fragment with a length of 320 nucleotides is cDNA derived from globulin. Globin cDNA can, however, be detected if globin RNA is less or less than 2-, 5,%. Of total RNA. If an RNA is present as isolated in too small a number of copies, to be able to be brought to this mode of analysis, it may be all purified by any of the RNA purification schemes known up to pfezenb until it represents 2 to 5% I tend to mix the RNA types that remain.

Example 3. Purification of the fragment of the uncleotidic sequence: about 550 base pairs in length, comprising a portion of the coding region for, 'HGS', is described together with a method for measuring the purity of the isolated sequence. It is shown that the purified fragment has a purity greater than 99%.

Purification of cDNA by human HCS

Placental polyadenylated RNA, isolated according to the procedure described in Example 1, was enriched in HCS mRNA by sedimentation in a 5% to 20% (wt. Volume) sucrose gradient solution at 4 ° C in a rotor of an ultracentrifuge at a speed of 25 000 rpm for 16 h. The 118 ... 143 gradient region was pooled and 100 µg of this RNA was used to synthesize a double-stranded cDNA, as described by Ullrich, A., Shine, J., Chirkwin, J., Pictet, R "Tischer, E., Rutter, WJ and Goodman, HM, Science, 196, 1313, 1977. The synthesis of the second side was stopped by extraction of the reaction mixture with a volume of ethanol at -'70 ° C. Hae III endonuclease cDNA digestion was performed in 50 μ 1 of 6 mM iris-HCl, pH = 7.5, 6 mM MgCh, 6 mM β -mercaptoethanol with two units of Hae III enzyme at 37 ° C, from 2 h; 0.1 units of bacterial alkaline phosphatase were added and digestion continued at 60 ° C for 10 min. The extraction followed by a volume of phenol-chloroform. DNA a. was precipitated with two volumes of ethanol at -70 ° C, dissolved in 20 μ 1 of 10 mM iris-HCl, pH = 8.1 mM EDTA and subjected to electrophoresis on a 6% polyacrylamide gel (w / v). Fig. 4 (F) shows the electrophoresis stain of the above reaction mixture, which reveals a prominent band corresponding to a nucleotide sequence of approximately 550 base pairs in length. The fragment of 550 base pairs in length was excised from gel and electrophoretically eluted with the results shown in fig. 4 (E).

The remaining material, which corresponds to a fragment of 550 base pairs, shown in FIG. 4 (E), was digested with 4 units of endonucleases Hha I in 50 μί of the same buffer used for digestion with Hae III at 37 ° C for 2 h. After extraction with phenol-chloroform and precipitation with ethanol. , the digestion products were separated by electrophoresis on a 6% polyacrylamide gel (w / v). The results are shown in fig. 4 (D).

The two fragments were electrophoretically eluted, combined and recombined by incubating in 20 µl of 66 mM tris-HC1, pH = 7.6, 6 mM MgCh, 15 mM dithiothreitol, 1 mM ATP containing 20 μg: ml The DNA was ligated at 15 ° C for 2 h. Then the reaction mixture was diluted to

two hundred 1 with 0.1 M NaCl, extracted with a volume of phenol-chloroform and DNA, precipitated with two volumes of ethanol. After resuspension in 20 μ1 of 10 mM tris-HC1, pH = 8.1 mM EDTA, the ligation products were separated by electrophoresis in a 6% (w / v) polyacrylamide gel. The result is shown in FIG. 1 (C). It can be seen from the electrophoresis stain on fig. 4 (C) that the two Hha I fragments were recombined in the original sequence with respect to each other, in order to reconstruct the nucleotide segment of 550. Thus, by ligation treatment, the 550 nucleotide fragment was reconstituted. Prior treatment with alkaline phosphatase ensures that the two Hha I fragments are re-assembled in the original sequence. The additional strips shown in FIG. 4 (C) resulted in dimer formation between Hha O fragments, because dimer formation is not hindered by alkaline phosphatase treatment.

The fragment reconstituted with 550 nucleotides was excised from the gel and electrophoretically eluted. The electrophoresis stain of the eluted material is shown in FIG. 4 (B). Fig. 4 (A) represents the stain of Hae III digested electrophoresis³²p of a two-sided DNA Ml3 DNA used as a size marker. Electrophoretic analyzes were conducted in a 6% polyacrylamide gel (w / v) in 50 mM frfs-borate, pH = 8.1 mM EDTA at 100 V for 2 h. After electrophoresis, the gel was dried and exposed to a film with fairies, to get a autoradiogram (or autoradiograms).

Purity of the reconstituted fragment of 550 nucleotides of HCS cDNA

Hae III fragments isolated from the reconstituted HCS cDNA were labeled / cu ³²p at the 5 'ends using polynucleotide kinase enzyme, obtained from the bacteriophage infected with Escherichia coli, by the method described in Panet, A. et al., Biochemistry, 12, 5045 of 4973. Then the fragment was digested with either Hha I or with Hpa II, in 50 μ1 of 6 mM fris-HCl, pH = 7.6, 6 mM MgCh, 6 mM β -mer'capttoethanol, at 37 ° C for 2 h. Then extraction with an equal volume of phenol followed. chloroform. DNA was precipitated with two volumes of ethanol at -70 ° C, resuspended in 20, 10 mM tris-HCl, pH = 7.6, 1 mM EDTA and subjected to electrophoresis, the gel was then exposed to a film. with X-rays to view the marked fragments, as described above.

78S07 '(5. The results, are «ă ^ atjgțe, on .fig. 5, Fig · SjJRf and, 5, (E) qeprezipt .dupiicâte • ăfe fragmentqior d © 550 nueleoides 4pa> inte-.4® epzimef digestion of reservoirs $ ş. jfȘg, .-. 5.:(C) .represip ^ p & fa, cgre sEZH-iţa from cliyâfjul © pjHJța I, .. and .fig. 5, (P) depreciation .pata .ore rgzpltă-din ₍clî, vajpbcu Hpftifl.

.Rurity of the brother of the 550 nuqleotidp ex jtyjgșufjțță. By the seriping of qUtqrădipgrăjpei 'RFQdpplui of clivpj and through, ςηηη / tificare,, distribribțtei^; qjad'ioacUvitate in TefipQre of the sweet digestions of endopupipases on restriction. Such measurements reveal the fact: the fragment: from Ήρ © î rjeqOn'șțitpit · from ADFfc from hcș nnwn. ' purified a'.fppt over 99% homogeneous.

4. Sftiezn «chicken plasmid. © ape nontine sqcyenfe_;&lt;^ KţJeoţ &lt;iţige. , of 550 pairs; encompassing the region's size, coding. for a HCS &lt;&amp; - a. prepare the 550 npclecftide fragment, cDNA of H £? Ș with a purity of more than 99% following the procedure · described in, &amp; χβιρρίρΐ 3. Terminal end 7, the phosphate was restored to a mixture of non-use containing - 50 mM fris-if Gl. more = r.8i5, ·. 10, mMÎ MșPl ?. E mM apermidine- 5 mM fi -mercaptpetenol., 3 t / »Weight / veium). gJiperoF 333 / PmPl AjPr 5 mpiteiu- of Ț4 ppiinneieo.tide work to a final yotpm of 40 cl Ja, 37 ° C for 4® 3 h. APN-nl was separated from one, reaction mixture by extraction, phenol, followed by precipitation. © tanpluiwji; Iapcfrerii · deUUCfeotizi synthetic: having the specificity of siinsnJui: of reification for, Rco RJ. and having the sequence 5'tCCQAATȚCGG.-3 ',, priswat according to Ό? χ. RA ,. Mc / ăodș in Enzymology, 12, 120, 1968, was then added to the HCS DNA in a molar ratio of approximately Jiy 50: 1, in 50 0: 1. die 66 mM fris-HCl, pH 7, .6;

9. m ^⁴ MgCU 15 mM dithiothreitol, 1 mM AȚR and 20 'g / ml D * DNA ligase. After mating at 4 ° C for 13 h.^re.actia + fpst stopped by phenol-qlorpfarm extraction · Ligation or, better said, the ligation product was precipitated with Otanpl, redissolved in 50ρ. 1 of 100 mM T4a, 50 mM fris-HCl, pH = 7.6; 7 mM fyfgGb and digested © with 50 units of endcoinction of Pco RI at 37 ° C timo dp 2 b. Digestion of cp endonucleases resulted in cleavage to the spindle, EcoRI of the decamers rearranged 'an increase in cDNA of HCȘ cp the cohesive ends of EcoRI, such as Sb cleavage of decanucleotide of unreacted ideas and of decanucleotides themselves. If the split chambers contain, of. that is, EcoRI terminates and competes with HCS cDNA for recombination with an elivaf-like plasmid, so with HCS cDNA it was isolated: gel electrophoresis bet before_:react with the transfer vector. Use of the .dfenBcteotidic rde / may lincfeer; up._;We have found that HCS cDNA can be> isolated as a fragment> from the plasmid in a form identical to that of the original fragment.

The transfer vector used was the bacterial plesmid! pMB-9 with a nuclear weight 3d> x, 10U of the xar molecule contains a single site - Eco .RE peepa-. xat as described by iRodriguez, RL and others rlGN-UCEA Șyrgpn ^ îcm <Qn Molecular and Genetic Biolagy Academic Press, New York, .1.95®, pp. 471 .., 477. Infection Έ. Sheets with rpMB * 9-Tetracycline-resistant GFP. The incorporation of the ASN into the RcoRI network of pMB-9 cannot affect the resistance: 1a tetracycline · or- OBicare other known properties of the plasmid. consequently, there are no -, - phenotypic differences between plasmid, recombinant and normal plasmids. Therefore, EgqRI ​​cuts pMB-9; being treated first with. : alcefline phosphatases; · According to me, the method described in^! detail in US Apudication 8Θ5 023: and Ullrfeh, A., Shine, Ji, Cfeârewin, J., flidtet, R "Tiscber, E., iRuîter, WJ and Goodman, i: M" Science 196, 1313, 197.7. Treatment with alkaline phosphatase ethylline 5 'EcoRI phosphates generated by plasmid and prevents plasmid self-binding gives DNA, ensuring this formation circle and, therefore, transformation is dependent on insertion of the DNA fragment containing terms: 5' host tissues. Alkaline phosphatase egg treatment was performed in a reaction mixture at a level of 0, enzyme units per milligram of plasmid DNA in 25 mM. frîs-iiCb = A, for 30 minutes, it has a temperature of 65 ° C, followed by the extraction with phenol, in order to ensure the elimination of the phosphatase and^: ethanol precipitation of DNA. Irrigation of HCS cDNA with pMB-9, treated as described, was carried out in 50 1 reaction containing 60 mM tris-HGl, pH = 8; 10 mM β -market-ptoethanol, 8 mM MqCb between 10 and 50 ng of purified HCS cDNA and about 500 ng of cleaved EcoRI · and 5 'dephosphorylated DNA plasmid. The reactions started with the addition of T4 ligase DNA at 5 μg / ml, allowing processing at 15 ° C for one hour, and thereafter diluted! to 0.25 ml with 120 mM NaCJ, 1 mM EDTA. The diluted reaction mixture was used directly for the transformation of E: sheets X-1776,

E: X-1776 sheets is the host body specially developed for the DNA recombination operation certified by

NIH as an EK-2 host. with directions, the US Federal Reserve's 78787 strain card is available from Roy Curtiss III, University of Alabama, Department of MicPobioIogy, Birmingham, Alabama. Bacteria were grown in 150 non-tritvv medium fed 100 µg / ml diaminopimelic acid (DAP) and 40 µg / ml thymine with a cell density of approximately 2χ, 10^κ cells / ml. Cells were harvested by centrifugation and washed 10 times in 60 ml of 10 mM NaGl, newly re-suspended and resuspended in 60 ml of transformation buffer containing 10 mM iris-HCl, pH = 8, 140 mM NaCl, 75 mM Bath. The suspended cell was kept on ice for 15 min, the cells collected by centrifugation and resuspended in 1.5 ml of the same transformation buffer. Suspended cells, 0.5 ml, were added to 0.25 ml of dilution ligation reaction mixture 20 and incubated on ice for 15 min, then transferred at 25 ° C for 4 min, then again on ice for 30 min. The 0.2 ml cell suspension was directly spread onto nutrient agar plates, aii-²⁵mented with 100 and 40 tg / ml thymine and 20 ml tetracycline. Four transformations were obtained, all containing 550 pairs of insertion bases that were released from the DNA plasmid by 30 digestion with either EcoRI or Hae III.

A transformed clone designated by pHCS-1 was collected for sequence analysis, E. coli X.1776-pHCS-1 grown in a convex nutrient medium. ³⁵The DNA plasmid was isolated from the acceptor and cleaved with EcoRI endonucleases. The product with 550 ba18 ζθ pairs was isolated from the linear product pMB-9 by electrophoresis in a 6% polyacrylamide gel._n Maxam and Gilbert procedure. Under HCS DNA fragments were prepared by incubation with restriction Hpa II endonucleases, and the 5 'terminus was labeled using polynucleotide kinase and gaimmaFP-ATP. Following the assay procedure, according to the Maxam pieduct, the DNA sequence of the donated HCS was determined; Compared to the known amino acid sequence of HCS, the 557 nucleotide sequence represents that portion of the HCS mRNA coding region of amino acids 24 to 191, plus 50 nucleotides of the 3 'untranslated region (Niall, HD, Hogan, ML, Sauer, R., Rosenblum, YY and Greenwood, FC, Proc. Nat. Acad. Sci. USA, 68, 866, 1971).

The primary structure of HCS mRNA, determined from the DNA sequence in the donated pHCS-1 fragment, is shown in Table 2, together with the amino acid sequence predicted therein, based on the known genetic code. The amino acid sequence determined from the nudeotide sequence is identical to the previously published amino acid sequences, first determined by chemical means. This shows that the HCS mRNA originally isolated was copied in vitro with high fidelity and that the HCS mRNA fragment was replicated with high fidelity in the transformed bacterium.

The nudeotide sequence of the HCS DNA side of p-HCS-1 is given in Table 2.

Table 2

W

<td>Wave </td><td>- Gin</td><td>- Thr</td><td>- Val</td><td>- Pro</td><td>- Leo</td><td>- Ser</td><td>- Arg</td><td colspan="4">Leu - Phe - Asp - η λ</td><td>His -</td><td>Ala -</td><td>Met -</td><td>Lion</td><td>-Glu</td>

<td>That </td><td>- His</td><td>- Arg</td><td>- Alas</td><td>- His</td><td>- Gin</td><td colspan="2">- Leu 5 '----</td><td></td><td colspan="3">Ala Ile Asp G GCCATTGAC</td><td>Thr ACC</td><td>Tyr TAC</td><td>Gin GAG</td><td>Glu GAGj</td><td>Phe TTT</td>

<td>Chou</td><td>Glu</td><td>Air</td><td>l'yr '</td><td>Ile</td><td>Pro</td><td>Lys</td><td>Asp</td><td>40 Gin</td><td>Lys</td><td>Tyr</td><td>Serum</td><td>Phe</td><td>Lion,</td><td>His</td><td>Asp</td><td>Serum</td>

<td>'AA'</td><td>Laat</td><td>\ CC</td><td>TAT</td><td>\ TC</td><td>THAT</td><td>\ AG</td><td>'-ACJ</td><td>GAG</td><td rowspan="2">AAG) is Thr</td><td>TAT</td><td>TOG</td><td>TTC</td><td>CTG</td><td>HOW</td><td>GAC</td><td>TCC</td>

<td>in the</td><td>Ίιε |</td><td>&gt; η</td><td>Het</td><td>Cysi</td><td>Asp</td><td>ssr</td><td>Ile</td><td>Pro</td><td>Pro</td><td>Serum</td><td>Asn</td><td>Met</td><td>Glu</td><td>Glu</td><td>Thr</td>

<td>CAC</td><td>The CC</td><td>TCQ</td><td>TTC</td><td>TCC</td><td>TTC</td><td>TCA</td><td>AAT</td><td>GCC</td><td>ACA</td><td>CCC</td><td>TCC</td><td>AAC</td><td>ATG 80</td><td>GAG.</td><td>GAA</td><td>ACC</td>

<td>Gin</td><td>Glro</td><td>IYS</td><td>Serum</td><td>Asn</td><td>Lion</td><td>Glu</td><td>Lion</td><td>Lion</td><td>Arg</td><td>Ile</td><td>Serum</td><td>Lion</td><td>Lion</td><td>Lion</td><td>Ile</td><td>Glu</td>

<td>CAA</td><td>CAG</td><td>AAA)</td><td>TCC</td><td>AAT</td><td>CTA</td><td>GAG</td><td>CTG</td><td>CTC</td><td>CGC</td><td>TRUMP</td><td>TCC</td><td>CTG</td><td>CTG</td><td>CTG</td><td>ATC 100</td><td>GAC</td>

<td>Serum</td><td>Trp</td><td>Lion</td><td>Gin</td><td>Pro</td><td>Wave</td><td>Arg</td><td>Phe</td><td>Lion</td><td>Arg</td><td>Serum</td><td>Met</td><td>Phe</td><td>That</td><td>So</td><td>Asn</td><td>Lion</td>

<td>TCG</td><td>TGG</td><td>CTG</td><td>GAC</td><td>CC</td><td>CTG</td><td>CGG</td><td>TTC</td><td>CTC</td><td>AGC</td><td>ACT</td><td>ATC</td><td>TTC</td><td>GCG</td><td>AAC</td><td>AAC</td><td>CTG</td>

<td>Wave</td><td>Tyr</td><td>Asp</td><td>Phe</td><td>Serum</td><td>Asp</td><td>Ηοι-</td><td>Asp</td><td>\ sp</td><td>Tyr</td><td>1 lys</td><td>Lion</td><td>Lion</td><td>L.ys</td><td>Asp</td><td>Lion</td><td>Glu</td>

<td>CTC</td><td>TAT</td><td>? q</td><td>ACC</td><td>TCG</td><td>GAC,</td><td>Α GC</td><td>HOW</td><td>GAC</td><td>TAT</td><td>CAC</td><td>1 TC</td><td>CTA</td><td>AAG</td><td>CAE</td><td>CTA</td><td>GAG</td>

20'

120

Cilii -îly b &lt;- 'Gin, Thr Leu Met Gly Arg Leu Glu Asp Gly Ser Arg Arg Thr

GAA GGC ATQ CAA ACG CTG ATG GCG AGG CTG GAA GAC GCC ACC CGC GGC ACT

140 . ' *·'

Gly Uliț be Leu 'Lyn Gin Thr Tyr Ser Lyn Phe Asp Thr Asn Ser His Asn

GGG GAC ATC CUG AAG GAG ACC TAC AGC AAG TIT GAC ACA AAC TCG CAC AAC

160 tis sp la 2U¹. I Lys Asn Tyr Gly Leu Leu Tyr Cyn Phe Arg Lys Asp

CAT AG: CA CTG CTO AAG AAC TAC GGG CTG CUC TAC TCC TTC ACG AAG CAC

180

Met-, Asp Lys Val Glu Thr Phe Leu, Arg Met Val, Gin Cys Arg. I'll be Val Glu

ATG GAC QQQ GTC GAG ACA TTC CTG CGO ATG GTG CAG TGC CGC TCT GTG GAG

191 '

Gly, Ser Cys Gly Phe

GGG AGG TGT GGC TTQ TAG GTGCCCGAGTAG CATCCTCTGACC CCTCCCCAGTGC CTCTCC TGCCC ---—- 3 '

The numbers refer to the starting amino acid sequences at the amino terminus. The sequence shown shows the sequence of the RNA sequence for HCS. except that U replaces T in mRNA. The amino acid sequence from positions 1 to 23 is also shown.

For &lt;a better understanding - of the invention, the following are also shown:

As a consequence of the known relationship of base pairs, which governs re- ₅splicing and transcription of DNA, isolation of mRNA containing the nucleotide sequence encoded for the aipino-acid sequence of the specific protein is equivalent to isolation of the same substance or gene, from the AON itself.

The mRNA data is transcribed from DNA to form complementary DNA, that is, cDNA, the DNA sequence is exactly reconstituted by it and ₁₅it may, by appropriate techniques, be inserted into the genetic material of another organism. The two complementary versions of the given sequence are therefore interconvertible and functionally equivalent to each other.

The nucleotide subunits of DNA and RNA - are linked or articulated together by phosphodiester linkages between the 5 'position of a · nucleoitidic sugar 25 and the 3' position of its next neighbor. Repeating such a link produces a linear polynucleotide having polarity in the sense hh, one end of which can be distinguished from the other._;j₀The 3 'end may have a free 3'-hydroxyl or the hydroxyl may be substituted with a more complex phosphate or citrate. The same is true for the 5 'end. In the organisms 35 eukaryotes! respectively those that have a defined nucleus and a mitotic apparatus, functional mRNA synthesis usually includes the addition of polyadenyl acid to the 3 'end of the mRNA. The messenger RNA can, therefore, be separated from other RNA classes isolated from a eukaryotic organism by means of cellulose column chromatography, to which the polytimidyl acyl is attached (Aviv, H. and Leder, P., 45

Proc. Nat. Acad. Sci. SU-.A., 69, 1408 - 1972). Other chromatographic methods exploiting the bases of pairs or,. more preferably, the affinity of these · bases of PIB-A for filler chromatography materials, containing dT.poh-U oligo, or combinations of poly-Ί and U-poly, for example, U-Sepharose poly, are also , convenient.

Reverse transcriptase catalyzes synthesis, complementary DNA in an RNA template, in the presence of the RNA template, a primer that can be a complementary polynucleotide oligo that has a 3'-hydroxyl and four itphosphate deoxynucleotides, dATP, dGTP, dGTF and dTTP. The reaction is initial by hecovalently associating this oligo-deoxynucleotide primer near the 3 'end of the RNA, followed by the step of adding the appropriate deoxynucleotides, determined by the reaction of the base pairs with the nucleotide sequence of the mRNA, at the 3' end of the chain. increase. The product molecule can be described as a hairpin structure in. which original RNA is paired by hydrogen bonding to a complementary strand of DNA, partially bent back over one of its ends. The DNA and RNA chains are not covalently linked to each other. The reverse transcriptase is also capable of catalyzing a similar reaction, using a single strand of DNA in the form of a hairpin, which has a loop of a single strand of DNA, which binds a set of ends (Efstrtiadis A. , Kafatos FC et al., Czech, 7.279.1976).

Restriction emdonucleases are enzymes capable of hydrolyzing DNA phosphodiester bonds, creating a disruption in the continuity of the DNA chain. Since the DNA is in the form of a closed loop, the loop is converted into a linear structure. The main aspect of the restriction enzyme is that its hydrolytic action is exerted only at a point where a nucleotide sequence appears. Such a sequence is defined as the restriction site for endonuclease restriction. The restriction endonucleases were isolated from a variety of sources and characterized by the nucleotide sequence after the sites of its restriction network. When acting on a double-stranded DNA, some restriction endonucleases hydrolyze the phosphodiester bond on both chains at the same point, producing hollow ends. Other catalysts hydrolyze the bonds separated by the few nucleotides from one to the other, producing regions with single chains at each end of the cleaved molecule. Such single-stranded ends are self-complementary, so cohesive, and can be used to re-hydrolyze DNA. Because any of the DNA, susceptible to cleavage by such an enzyme, must contain the same recognition site, the same cohesive ends will occur, as well. that it is possible to combine heterogeneous DNA sequences that have been treated with restriction endonucleases from other similarly treated sequences (Roberts RJ, Crit. Rev. Biochem., 4,

123,. 1976).

It was observed that the reaction sites for a given enzyme are relatively rare and unevenly distributed. Regardless of why a restrictive site exists within a given segment, this is a problem that needs to be determined empirically. Moreover, there is a large and growing number of restriction endonucleases isolated from a variety of sources with a variety of site specificity, so that there is a reasonable probability that a segment of one thousand nucleotides will contain one or more many restriction sites.

The microorganism modified to contain codons, for human chorionic somatomamotropin, comprises the following nucleotide sequence: 5'-G GCLp. ATMs GAKw ACL * TAX CAJ29 GA Jan TTKat GAJ.% GAJ-a ACL34 TAKafe ATW CCIot AAJas GAKsi) CAJw AAȚi TAK «QRu S» a TTRu Xc TY «CAK, - GAKv QR /, 8 S« CAJ / ,, i ACLsoQRsi Ssi TTKkTGKss TTKaiQRw S55 ATMs * QR57 Sv ATMssCCLsg ACLso CCLîi QR-p S52 AAK «3 ATGGAJss GAJ« 6 ACLtf CA.Ls CAJcj AAJ70 QR71 S71 ΛΑΚ; .- X73 TY73 GAJ74 X75 TY75 X76 TY₇6 W77 GZ77 ATM78 QR79 S79 Xso ΤΥβι) ΧδΙ TY₈₁ Xsa ΤΥβ2 ATMîa GAJe-, QR35 Sas TGGXst Ί Y₈₇

CAGES OCLsn GTLon W_9l GZ₉j TTK.92 Xa TYra W₉₄ GZsi QR95 Sos ATCTTK97 GCLgs AAK99 AAKioo X101 TYjoi GTIjioa TAK103 GAK104ACL106 QRioeSios GAK107 QRios Sioe GAJ109 GAKnoTAKiu CAKnz X113Y113 X114 TY11V AAJus GAK116 TY7

GAJnsGAJuo GCLno ATM® CAJisa ACL-m

X.L24 TY124 ATGGGLui W „27 GZ127 Xl28 ΊΥ128 GAJ329 GAKl30 CGLl31 QR132 S132 Wttt GZw W134 GZiM ACL135 GGL, 36 GAJ1117 ATM, 138 X139 TY139 AAJl40 CAJ141 ACL1444 TAKw QR 47 Xl56 TYlS6 Xl57 TY157 'AAJ_15S XU3 TYlfe AAK159 Tyle Xl62 TAK16O TAK164TGK105TTK166 GGL10I GZ167 AAJ168 I GAK169 ATGGAKlT Wl67, X177 TTKl76 AAJl72 TYl77 GTL173 Wl78 GAJt74 GZl78 ACLl75 CAJlSI ATGGTLlSO GTLl85 TGK182 GAjl86 Wl83 GGL.I87 GZl83 QR188 QRl84 Sl84 Sl-88 TGKl89 GCGCGA TAGGTK TTKl91 GTAGCiA GGLl90 TCjCTGT / GACrCG TQCGCA GTCCCT CTCCGGi CC-Q 'wherein A is deoxyadenyl, G is deoxyguanyl, C is deoxycytosil. T is Limidil, J is A or G, K is T or C_; L is A, TC or G? M is A, C or T; Xn is T or C, if y «is A or G and C, if y« is C or T, · y «is A, G, C or T, if x-ι is C and A or G, if x» is T; Wn is C or A, if Z »is G or A and C, if Zn is C or T, Zn is A, G, C or T, if Wn is C and A or G, if Wn is A; QRn is TC, if Sn is A, G, C or T and AG, if Sn is T or C; Sn is A, G, C or T, if QRn is TC and T or C, if QRn is AG, and the numbers subscribed, n refers to the amino position of the acid in human chorionic somatomamotropin, for which the nucleotide sequence corresponds, according to the genetic code , the amino positions of the acid being counted from the amino end.

In the following, the following data is given to explain the invention.

A novel process for purifying a desired nucleotide sequence of complementary DNA with an individual cDNA species is described in the present invention. The method utilizes the restriction endonuclease cleavage of a cDNA transcribed from a complex mixture of mRNA. The process does not require excessive purification of the RNA, but instead uses RNA transcription in cDNA, fragmentation of the specific sequence of this cDNA with one or two restriction endonucleases and fractionation of restriction cDNA fragments based on their length. The use of restriction endonucleases eliminates size heterogeneity and produces homogeneously sized DNA fragments from any cDNA species containing at least two restriction sites. From the heterogeneous initial population of the transcript cDNA, fragments of uniform size from the desired sequence are produced. The fragments may be several hundred nucleotides in length and may, in some cases, include the entire structural gene for the desired protein. The length of the desired fragments depends on the number of nucleotides that separate the restriction networks and will usually be different for the different regions of the DNA. Fractionation in length allows the purification of homogeneous populations of fragments having the desired sequence. The fragments will be homogeneous in size and of high purity in terms of nucleotide sequence. Current separation methods and analyzes allow isolation of such fragments from mRNA-corresponding species that represent at least 2% of the mass of the transcribed RNA. The use of the previous methods of RNA fractionation for the prepurification of mRNA prior to transcription will result in the decrease of the lower real detection limit of less than 2% of the total mRNA isolated from the body.

The specific sequences purified by the procedure outlined above may be further purified by a second specific cleavage with a restriction endonuclease, capable of cleavage, of the desired sequence at an internal site. This cleavage is reduced to the formation of two subfragments of the desired sequence, separable based on their length. The sub-fragments feel separated from the unclipped and specifically cleaved sequences of contamination, which have the same original size. The method is based on the rarity and placement of the site, the sources of recognition of restriction endoinucleases that result in an extremely low probability that a contaminated substance of the same original length will be cleaved by the same enzyme to result in fragments of the same length as that of the desired sequence. After the separation of contaminants, the sub-fragments of the desired sequence are not to be re-used, using the techniques known in this field, to reconstruct the original sequence. A method by which the sub-fragments can be combined with each other only in the correct order is described. The two sub-fragments may be prevented from merging together in the reverse order of their original sequence.

Different variations can be used ²⁴ of the above-mentioned methods in combination with the appropriate marking techniques to obtain precise quantitative measurements of the purity of the isolated sequences. Isolated techniques can be applied to produce the nucleotide sequence with a purity greater than 99%.

The cDNA isolated and purified by the methods described can be recombined with a convenient transfer vector and transferred to a host microorganism by known methods. New plasmids containing nucleotide sequences encoding most of the HCS portion were produced. The techniques described can be used to isolate and purify human growth hormones from other animal species and to reconstruct new transfer vectors and microorganisms containing these species. The new plasmids contain nucleotide sequences that encode most portions of human chorionic somatomotropin.

The present invention uses as a polyadenylated starting material a crude or partially purified mRNA, which may be heterogeneous in sequence and of molecular size. The selectivity of producing RNA isolation is enriched by any method that results in an enrichment of the desired mRNA in the heterodispersed population of the isolated mRNA. Any such purification method may be used in connection with the method of the present invention, the method provided by not introducing the endonucleotide cleavage of the mRNA. An important consideration is the selection of an appropriate tissue source for the desired mRNA. Often, this choice will be dictated by the fact that the protein to be obtained is prepared only by a certain specialized tissue of a differentiated organism. This is the case, for example, with non-optic hormones, such as the HCS hormone. In these cases, it is discovered that a variety of cell types or microbial species may serve as a source for the desired mRNA. In these cases, preliminary experimentation will be required in order to determine the optimal source. Frequently, it is found that the desired mRNA ratio may be / increased by the cell's response to stimuli of the environment. For example, hormone treatment may cause the desired mRNA production to increase. Other techniques include raising it at a particular temperature and exuding it to a specific nutrient or other chemical.

Pre-purification in order to enrich the desired mRNA sequences can also be performed by using conventional methods for RNA fractionation, after isolation, from the cell. Anything . A technique that does not result in RNA degradation can be used. Preparative sedimentation techniques, in a sucrose gradient; and gel electrophoresis are special. convenient.

The mRNA must be isolated from the source cells under conditions that exclude mRNA degradation. The action of RNAi enzymes is particularly convenient to avoid, because these enzymes are capable of hydrolytic cleavage of the nucleotide sequence of zVRN. ; Hydrolysis of a link in the sequence results in the breaking of that sequence and the loss of the RNA fragment which contains the original 5'-end of the sequence. A convenient method for inhibiting RNA during cell extraction is described in: literature (US Patent Application: 805023). The method develops the use of 4M guanldine thiocyanate and 1 M mercaptoethanol during the cell break-up phase. In addition, a low temperature and a pH close to 15.0 are helpful in further reducing and degrading RNA isolated by RNAase.

Before applying the method according to the present invention, mRNA must be prepared essentially without any contamination with protein, polysaccharides and lipids. Standard methods are known in the art for such purification. The RNA thus isolated contains both messenger RNA and non-messenger RNA. A convenient method for 'separating RNA from eukaryotes is column chromatography of oligo-dT cellulose or other materials, column with substituted oligonucleotides, such as poh-U-Sepharbza, having the advantage of hydrogen bond specificity conferred by the presence of polar acid' enilic, at the 3 'end of eukaryotic mRNA.

Initial stage. din. The process of the present invention is the formation of complementary DNA with isolated heterogeneous mRNA sequences. The enzymes chosen for these reactions are reverse transcriptases, although, in principle, any enzyme is capable of forming a complementary DNA a faithful copy of the usable mRNA. The reaction may be carried out under the conditions known in the art, using mRNA as a template and a mixture for or as precursors for the DNA chain, a mixture consisting of four triphosphate deoxynucleosides dATP, dGTP, dCTP and dTTP. It is convenient to provide that one of the deoxynucleoside triphosphates is labeled with a radioisotope, e.g.³²p in position «, in order to conduct the operation course, in order to achieve, the recovery of the product, according to the current procedures of separation, as, is the chromatography and electr of the ore and with the purpose of making, quantitative determinations in, · what is regards the recovered part (Efstratiadis A., Kafatos FC, Maxam AM, Maniatis T "Cell. 7, 279, 1976).

The triancribed cDNAs, produced by the reverse transcriptase reaction, are somewhat heterogeneous with respect to the 5 'end and 3' end sequence due to the variation in the initial and end points. of individual transcripts, compared to the mRNA model. The variability at &lt;5 'end is due to the fact that the primary oligo-dT, used to initiate synthesis, is capable of binding, in a variety of locations along the polyadenylated mRNA region. The synthesis of cDNA, transcribed begins at an intermediate point - from the poly-A region and a poly-A of variable length is transcribed according to the site, which initially binds the oligo-dT primer. It is possible to avoid this indefiniteness by using a · primer · which, in addition to an oligo-dT tnact, contains one or two nucleotides of the RNA sequence itself, through which a primer is produced will be preferred and will have a binding site defined for initiating the transcription reaction.

The non-determination at the 3 'end of the transcribed DNA is due to a variety of factors that affect the reverse transcriptase reaction and the possibility of partial degradation of the RNA template. The isolation of the transcribed, specific cDNAs of maximum 'length' is greatly facilitated, but the conditions for the reverse transcriptase reaction are chosen in such a way that they not only promote 'lengths, synthesis, but also' to suppress the synthesis of cDNA mid-chains. ' Preferred conditions for reverse transcriptase of avian myeloblastosis virus are not given in the examples. The specific parameters that can be varied to achieve the maximum production of high fidelity, long-chain transcribed DNAs are: reaction temperature, concentration, salt, enzyme quantity, initiator concentration versus template, and reaction time. .

The temperature conditions and the salt concentration are chosen so as to optimize the coupling of the specific bases between the oligo-dT initiator and the polyadenylated portion of the RNA template. Under the correctly chosen conditions, the primer will be able to bind to the polyadenylated region of the RNA template, but no specific initiation will occur due to its binding to other locations of the template, in short, sequences rich in A will be prevented in a manner. substantial. The effects of temperature and salt concentration are interdependent. As the temperatures are higher and lower, the salt concentration decreases the stability of the specific coupling interaction at the bases. The reaction time is kept as short as possible in order to prevent the initiation of non-specifics¹ and to minimize the opportunity for degradation. Reaction times are interrelated with temperature, and low temperatures require longer reaction times. At a temperature of 42 ° C, reaction times vary from 1 to 10 minutes and are convenient. The primer will be present in a molar excess of 50 ... 500 over the RNA template, so the enzymes will be present in a molar excess similar to the RNA template. The use of excess enzymes and pnimer allows the initiation and growth of the cDNA chain, thus producing long-chain transcribed cDNAs and this production takes place efficiently at short incubation times.

In many cases, it is possible to perform the remainder of the purification process of the present invention using single-stranded cDNA transcripts. However, as will be shown below, there may be cases where the desired restriction enzyme is one that acts only on double-stranded DNA. In these cases, the cDNA prepared as described above can be used as a template for the synthesis of a two-stranded DNA, using a DNA polymerase, such as a reverse transcriptase and a nuclease capable of hydrolyzing a DNA. with one side. Methods for preparing two-sided DNA in this manner have been described in the literature (Ulrich, A. and Others, Science, 196, 1313, 1977).

Heterogeneous DNA, prepared by the transcription of heterogeneous mRNA sequences, is then treated with one or two restriction hemonucleases. The choice of endonucleases to be used depends, first, on the prior determination of recognition sites for the enzymes that exist in the cDNA sequence to be isolated. The method depends on the existence of two such sites. If the sites are identical, a single shot will be enough for me. The desired sequence will be cleaved at both sites, eliminating the heterogeneity of dimensions as much as possible and creating a population of molecules called fragments, which contain the desired sequence and which are homogeneous in length. If the restriction networks are different, two enzymes will be needed in order to produce fragments of the desired homogeneous length.

The choice of the enzyme or restriction enzymes capable of producing a nucleotide sequence fragment of the desired or, rather, optimal length, encoding all or a portion of the desired protein, must be empirically performed. If the amino acid sequence of the desired protein is known, it is possible to compare the nucleotide sequence of the nucleotide fragments of uniform length produced by the cleavage of restriction endonucleases, with an amino acid sequence, for which its codes, using the known common genetic code relationship for all life forms, means living organism. A complete amino acid sequence for the desired protein is not required, because identification with reasonable accuracy can be performed based on a partial sequence. Where the amino acid sequence of the desired protein is not known, the uniform length of polynucleotides produced by cleavage of restriction endonucleotides may be used as evidence capable of identifying the desired protein synthesis in a suitable in vitro protein synthesis system. As a variant, mRNA can be purified by chromatographic affinity. Other techniques that may be suggested to those skilled in the art will be those suitable for this purpose. The number of restriction enzymes convenient for use depends on the type of cDNA that is used, whether it is single-sided or two-sided. Preferred enzymes are those capable of acting on single-stranded DNA, which is the product of the immediate reverse transcription of mRNA. The number of restriction enzymes, now known, to be able to act on single-stranded DNA, is limited. The enzymes Hae III, Hha I and Hin (f) I are currently known to be capable. In addition, Mbo II enzymes can act on single-stranded DNA. Where studies show that restriction enzymes can act on one-sided DNA, such enzymes may indeed be included in the lysate of preferred enzymes. Suitable additional enzymes include those specified for two-sided cDNA. Such enzymes are not. preferred, because they are: necessary for the additional reaction in order to produce cDNA with two; sides, realizing, increasing the opportunity: for the loss of long sequences and for other losses due to the incomplete recovery. The use of a cDNA with two sides has additional technical disadvantages, as sequence analyzes feel much more complex and laborious. For these reasons, single-stranded cDNA is preferred, but two-stranded cDNA is also applicable.

, The cDNA prepared for the treatment of restriction endoniucleases may be. radioactive labeled, so that it can be detected after the following steps. One technique: preferred is that of incorporating a radioactive ion, such as / for example,³²p in the position. · «· of one of the four deoxyphiincleoside triphosphates. precursors. Activity: the highest is obtained when the concentration of the radioactive precursor is relatively high relative to the deconcentration of the non-radioactive form. With all these, the total concentration of any triphosphate. The deoxynucleoside will be larger than 30 μM, in order to maximize: the length of the cDNA obtained in the reverse transcriptase reaction (Efstraitiadis, A. et al., Cell, 4, 367, 1975). In order to determine the 'nucleotide sequence of the cDNA, the 5' ends may be conveniently labeled ou ^ .p in the catalyzed reaction concerning the polynucleotide enzyme kinase (Maxam and Gilbert, W., Proc. Niat'l Acad. Sci USA, 7-4, 560 of 1977).

/ Fragments that have been produced by the action of the restriction enzyme or the combination of two restriction enzymes may be separated from each other and the heterodispersed sequences, due to the stasis of recognition by an appropriate technique, capable of polynucleotide separation, based on differences in length. Such methods include a variety of electrophoretic and sedimentation techniques using an ultra-centrifugal technique. Gel electrophoresis are preferred, as they best achieve the resolution based on the length of the polynucleotide, in addition, the method readily allows for the quantitative recovery of the separated materials. Suitable gel electrophoresis methods have been described in the literature (D.mgman, GW and Peacok, AC, Biochemistry, 7, 659, 1958 and Jeffrey, A., Vande Sande, H., Biochemistry, 14, 3787, 1975). Prior to the treatment with the transcribed cDNA restriction endonucleases, obtained from most sources, it is found that they are heterozygous: in size, respectively in: length. By / by the action of properly selected restriction endonucleases or pairs of 'endonucleases, the polynucleotide chain, which contains the desired sequence, will be cleaved at the respective restriction site, in order to obtain polynucleotide fragments of uniform length. After gel electrophoresis, they will be observed to form a distinct band / band. Depending on the presence - or absence of stasis¹of restriction on other sequences, other discrete bands may be formed, which will be similar-like, but of different lengths in front. of the desired ·· sequence. Thus, as a consequence of the action of the restriction endonuclease, the electrophoresis spike will reveal the occurrence of one or more discrete bands, while the remaining cDNA will continue to be heter.odispersal. If the desired cDNA sequence comprises the majority of the present polynucleotide species, the stain of electrophoresis will reveal the majority of the cDNA present in the ... band. chalk diss.

Although it does not hesitate: desirable as two sequences d.^: Yesterday you got to be cleaved by restrictive enzymes to get fragments of absolutely the same length, a method for determining. - the purity of the fragments of defined length is desired. The sequence analysis of the electrophoresis band can be used to detect impurities representing 10% or more of the in-band immaterial. A method for detecting lower levels of impurities has been developed as part of the present invention, discovered according to the general principles applied in the initial method of isolation. The method imposes a fragment of the nucleotide sequence. It must contain a recognition site for the restriction endonucleases that are not used in the original work. Treatment of the immaterial nucleotide-d or, better, of the polynucleotide eluted from the electrophoresis gel band, with restriction endonucleases capable of acting internally on the desired sequence, will have aa: result in the cleavage of the desired sequence into two subfragments, many, probably, by uneven length. These sub-fragments after electrophoresis will form two or more discrete bands at positions corresponding to the respective lengths, the sum of which is equal to the length of the polynucleotide before cleavage. Contaminants in the initial or original band are not susceptible to the restriction enzyme and can be expected to migrate to the original position. Contaminants containing one or more si78407

SI enzyme recognition cushions can be expected to result in two or more subfragments. Because the distribution of recognition sites is thought to be essentially random, the probability of a contaminant giving a subfragment of the same size as that of the desired sequence is extremely low. The amount of material present in any band of the radioactive labeled polynucleotide may be determined by quantitative measurements of the amount of radioactivity present in each band or by any other appropriate method. A quantitative measure of the purity of the fragments of the desired sequence can be obtained by comparing the relative quantities of material present in these bands, which represent subfragments of the increased sequence with the total amount of material.

Following the separation above, the desired sequence can be reconstituted. DNA ligase enzyme that catalyzes end-to-end binding of DNA fragments may be used for this purpose. The electrophoresis gel bands representing the subfragments of the desired sequence can be eluted separately and combined in the presence of DNA ligase, under appropriate conditions (Scaramela, V. et al., Proc. Nat'l Acad. Sci, USA, 67, 1468, 1970), where the sequences to be joined are not with the fused ends, and the ligase contained in E. coli (Modrichi, P. and others, J. Biol. Chem., 245) can be used. , 3626, 1970).

The efficiency of reconstruction of the original sequence from the subfragments produced by the treatment with restriction endonucleases will be greatly enhanced by using the method to prevent improper reconstitution in the sequence. This undesirable result is prevented by the treatment of the homogeneous length fragment of the desired cDNA sequence, with an agent capable of removing the 5'-terminal phosphatic groups from the cDNA, before the homogeneous cDNA cleavage with a restriction endonuclease. The enzyme, alkaline phosphatase, is preferred. The 5 'phosphatic terminal groups are structurally necessary for the subsequent inhibition of DNA ligase used to reconstitute cleaved subfragmenites. Therefore, the ends having a 5 'phosphate terminal cannot be covalently bound. The DNA subfragments can be joined only to the ends containing 5'-phosphate generated by the cleaved restriction endonucleases described in detail in the literature (Patent Application, USA, no. 805 023).

Most of the cDNAs transcribed under the conditions used are derived from the mRNA region, which contains the 5 'end of the model mRNA by specific priming on the same template with a fragment obtained by restriction endonuclease cleavage. In this way, the method described above can be used to obtain not only the fragments of the specific nucleotide sequence related to the desired protein, but also to the entire nucleotide sequence encoding the protein of interest.

The purification process is of particular significance in the deconvolution of human genes, which, according to the rules or, more properly, the US federal laws, can only be put into recombinant DNA and then into the bacterium afterwards, which genes have been very carefully purified. or if the experiments were carried out in special installations with high risk (P4J / 12 / (Federal Register, vol. 41, no. 131, of July 7, 1967, pp. 27 902—27 943). The present method allowed the production of sufficiently pure human genes, comprising most of the HCS structure. Human genetic material, isolated and purified as described above, may be incorporated into recombinant plasmids or other transfer vectors. Chemically synthesized double-stranded oligonucleotide liners containing the restriction endonuclease recognition sequence may be attached to the isolated cDNA ends to facilitate further enzymatic removal of the human gene portion from the DNA transfer vector ( Scheler, RH et al., Science, 196, 177, 1977). The DNA transfer vector is converted from a continuous loop to a linear form by treatment with an appropriate restriction endonuclease. The ends lined by this are treated with alkaline phosphatase, to remove the 5 'terminal phosphatase groups, thus preventing the DNA transfer vector from being able to form a continuous loop again in the DNA ligase reaction, without first incorporating a segment of human DNA. The cDNA with the oligonucleotide lynx and the die transfer vector of the treated DNA were mixed together with the DNA-ligase enzyme to combine the cDNA with the DNA vector. forming a continuous loop of the recombinant vector having cDNA incorporated therein. Where the transfer vector plasmid is used, the closed loop will usually be the only form capable of transforming the bacteria. Transformation, as understood in the art, is the term used to denote the process by which a microorganism incorporates extracellular DNA into its own genetic constitution. The plasmid DNA in the form of a closed loop may thus be incorporated under the conditions of the appropriate environment. The closed plasmid loop, embedded, follows replication in the transformed cell, and the replicated copies are distributed to produce cells, when cell division occurs. As a result, a new cell line is established, which contains the plasmid and transmits its genetic determination. Plasmid transformation in this way, in which plasmid genes are maintained in the cell line by plasmid replication, occurs at very high frequencies, when DNA plasmid transformation is in the form of a closed loop and does not occur or rarely occurs, if the linear plasmid DNA is used. Once a recombinant transfer vector has been created, the transformation of a convenient microorganism is a forward-looking process, and the new microorganism, which contains human genes, can be easily isolated, using appropriate selection techniques that are known in the art.

The invention has the following advantages:

- allows the isolation and purification of HCS nucleotide sequences, even when they are present with a frequency of only 2% of a heterogeneous population in mRNA sequences;

- combined with other known mRNA fractionation processes, the process can be used to isolate and purify the specific sequences present even at very low frequencies in the total RNA population;

- the process is generally applicable to mRNA species extracted from any virtual organisms, thus representing a powerful tool for the basic production of proteins that have medical, commercial or research interests and in useful quantities;

- performs a nucleotide sequence that purifies for human HCS with a purity of over 99%;

- Creates a microorganism that produces HCS.