Method of cdna fragment cleaning with specific nucleotide sequence

The invention relates to a method for purifying a fragment of a cDNA having a specific deoxyribonucleotide sequence and suitable for recombination with a DNA transfer vector and transformation into a microorganism, starting from a population of cDNAs of heterogeneous length and sequence, wherein the fragment to be purified is a minority of this population and at least a portion of the cDNAs has at least two restriction sites and wherein the population of cDNAs is produced using a reverse transcriptase catalyzed reaction from a population of mRNA of heterogeneous length and sequence. This method is according to the invention characterized in that

a) the population of cDNAs at the restriction sites by incubating a reaction mixture containing this population of cDNAs and a restriction endonuclease specific for these restriction sites, for example by incubating this population with Haelll and / or Hhal at a pH of 7.5 and hydrolyzed enzymatically to fragments of the cDNAs at 37 ° C. for 1 to 2 h ,

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- 7 b) the fragments of the cDNAs are fractionated according to their length, for example by gel electrophoresis, and the fragments of the cDNAs containing fragments of the CNSs containing homogeneous length are separated therefrom and

c) captures the fraction which predominantly contains the cDNA fragment having the desired specific deoxynucleotide sequence, said fragment having a specific deoxynucleotide sequence and containing a restriction site of the cDNA useful for recombination with a DSN transfer vector and for transformation into a microorganism by further purification that fraction of homogeneous length cDNA fragments, which predominantly contains a cDNA fragment having a specific deoxynucleotide sequence,

d) is hydrolyzed to terminal 5'-phosphate groups of these cDNA fragments by incubating these fragments with alkaline phosphatase, preferably at a pH of 7.5 and at 60 ° C for 10 minutes, and

e) the cDNA fragments at the restriction site by incubating a reaction mixture containing these fragments and a restriction endonuclease-specific reaction mixture, for example by incubating these fragments with Hhal or Hpall or PvuII at a pI value of 7.6 and at 37 ° C for 2 h, are hydrolyzed enzymatically to sub-fragments of these fragments,

f) the resulting sub-fragments are fractionated according to their length, for example by gel electrophoresis,

g) capturing the fractions containing the two subfragments of the desired specific deoxynucleotide sequence, hl the subfragments by incubating a DNA ligase, ATP and the reaction mixture containing the two subfragments, for example by incubating the DNA ligase, the sub Fragments and ATP with a p ^ value of 7.6 and at 15 ° C for 2 h, are enzymatically and covalently recombined to a cDNA fragment having a specific deoxynucleotide sequence, and

i) fractionating the ligase-treated cDNAs by, for example, gel electrophoresis, and capturing that fraction which contains a cDNA fragment which displays a specific deoxynucleotide sequence and is substantially free of contaminating cDNA sequences.

The invention thus provides a method by which nucleotide sequences can be isolated and purified which may be present at a frequency of only 2% in a heterogeneous population of mRNA sequences. The method can be further combined with known methods for fractionating mRNA to isolate and purify sequences present in the entire RNA population at even lower frequencies than after initial isolation. The method is generally applicable to mRNA from virtually any organism and therefore provides the effective means to produce suitable quantities of such proteins of interest or interest to the research. The method does not require extensive purification of RNA, but instead makes use of transcription of RNA into cDNA, sequence-specific fragmentation of this cDNA with one or two restriction endonucleases and fractionation of the cDNA restriction fragments by their length. The use of restriction endonucleases eliminates size differences and results in DNA fragments of homogeneous length from fragments of any cDNA containing at least two restriction sites. From the initially heterogeneous population of cDNA transcripts uniformly large fragments of desired sequence arise. The fragments may be several hundred nucleotides in length and occasionally contain the entire structural gene of the desired protein. The length of the fragments depends on the number of nucleotides lying between the restriction sites and is usually different in different regions of the DNA. The length fractionation allows purification of a homogeneous population of fragments of desired sequence. The fragments are of homogeneous size and highly pure in nucleotide sequence. Current separation and analysis methods allow the isolation of these fragments of the mRNA concerned, which accounts for at least 2% of the total RNA. The use of the known RNA fractionating No. 369386

The pre-transcription pre-purification procedure reduces the true lower detection limit to less than 2% of total mRNA isolated from the organism.

Special sequences purified by the above method can be further purified in a second specific cleavage reaction with a restriction endonuclease which cleaves the sequence at one site in the interior. This cleavage leads to two subfragments of desired sequence, which can be separated due to their length. The subfragments are separated from uncleaved and specifically cleaved contaminating sequences of substantially starting size. This method relies on the rarity and random arrangement of the restriction endonuclease recognition sites, making it extremely unlikely that a contaminating portion of equal length will be cleaved from the same enzyme to form fragments of the same length as the desired sequence , After separation from the contaminants, the subfragments of the desired sequence can be assembled by known methods to reconstitute the starting sequence. However, the two subfragments must be prevented from linking in the reverse order of their source sequence. The method according to the invention allows subfragments to be linked only in the desired order.

Modifications of the outlined methods may be used in combination with appropriate labeling techniques to allow accurate quantitative measurements of the purity of the isolated sequences. These combined techniques were used to prepare a known nucleotide sequence of greater than 99% purity.

The cDNA isolated and purified by the methods described above can be recombined with a suitable transfer vector and transferred in a known manner into a suitable host microorganism. New plasmids have been generated which contain the nucleotide sequences encoding the major part of the human chorionic somatomammotropin or human growth hormone, respectively. In addition, new microorganisms have been generated that contain most of HCS or HGH as part of their genetic makeup. The disclosed methods can be used to isolate and purify growth hormones from other animals and to produce novel transfer vectors and microorganisms containing these genes.

The process according to the invention uses as starting material polyadenylated, crude or partially purified mRNA which may be heterogeneous in sequence and molecule size. The selectivity of the RNA isolation procedure is increased by any method that results in enrichment of the desired mRNA in the heterodisperse population of the isolated mRNA. Any pre-purification of this type can be combined with the method according to the invention, provided that the method in question does not cause endonucleolytic cleavage of the mRNA. An important initial consideration is the choice of the appropriate tissue for the desired mRNA. This choice is often determined by the fact that the final protein to be produced is only produced by a particular specialized tissue of a differentiated organism. This is the case, for example, with the peptide hormones such as growth hormone or HCS. In other cases, different cell types or microbial species may serve as a source of the desired mRNA. In these cases, some preliminary tests to determine the optimal starting material are required. It is often found that the amount of desired mRNA can be increased by taking advantage of the cell's response to environmental stimuli. So can eg treatment with a hormone will cause increased production of the desired mRNA. Other methods use growth at a certain temperature or in the presence of a particular nutrient medium or other chemical substance.

The pre-purification to enrich the desired mRNA sequences can also be carried out using conventional methods for fractionating the RNA after its isolation from the cell. Any method that does not degrade the RNA can be used. The methods of preparative sedimentation in a sucrose gradient and gel electrophoresis are particularly suitable.

The mRNA must be isolated from the source line under determinations which preclude its degradation. In particular, the effect of RNase enzymes must be prevented because these enzymes

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- 9 are capable of hydrolytic cleavage of the RNA nucleotide sequence. Hydrolysis of a bond in the sequence results in the disruption of this sequence and loss of the RNA fragment containing the original 5 'end of the sequence. A suitable method for inhibiting RNase during extraction from cells is disclosed in DE-OS 2822568. During this process, 4 molar guanidinium thiocyanate and 1 molar mercaptoethanol are used during the disruption of the cells. Furthermore, low temperatures and p ^ values ​​close to 5.0 are helpful in reducing RNase degradation of the isolated RNA.

Prior to application of the method of the invention, a mRNA substantially free of contaminating protein, DNA, polysaccharides and lipids must be prepared. To carry out this cleaning, standard methods are available. The isolated RNA contains both nonmessenger RNA and messenger RNA. A convenient method of separating eucaryotic mRNAs is by chromatography on oligo-dT cellulose or other oligonucleotide substituted column material such as poly-U-Sepharose using the specificity of hydrogen bonding, which depends on the presence of polyadenylic acid on the 3 'End of the eucaryotic mRNA decreases.

The first step of the method of the invention is the formation of the DSN which is complementary to the isolated heterologous mRNA sequences. The enzyme of choice in this reaction is reverse transcriptase, although in principle any other enzyme could be used which can produce a genuinely complementary DSN copy of the mRNA template. The reaction can be carried out under the conditions known from the literature, using mRNA as template and a mixture of the deoxynucleoside triphosphates dATP, dGTP, dCTP and dTTP as precursors for the DNA strand. Suitably, one of the deoxynucleoside triphosphates with a radioisotope, eg³³P, marked in α-position, to follow the course of the reaction and the work-up and separation, for example by chromatography and electrophoresis, to control and make quantitative information; see. Efstratiadis, Α., Et al, loc. Cit.

The cDNA transcripts obtained in the reverse transcriptase reaction are somewhat heterogeneous with respect to the 5'-end and 3'-end sequences because of different starting and ending points of the individual transcripts relative to the mRNA template. The variation possibilities at the 5 'end are presumably due to the fact that the oligo-dT primer used to initiate the synthesis is capable of binding at numerous sites along the polyadenylated region of the mRNA. The synthesis of the cDNA transcript begins at an indeterminate point in the poly A region, then a variable length of the poly A region is transcribed, depending on the initial binding site of the oligo dT primer. One can avoid this ambiguity by using a primer which, in addition to the oligo-dT tract, contains one or two nucleotides of the RNA sequence itself, thereby obtaining a primer which has a preferred and defined binding site for initiating transcription. The uncertainty at the 3 'end of the cDNA transcript is due to several factors affecting the reverse transcriptase response and the possibility of partial RNA template degradation. The isolation of specific maximum length cDNA transcripts is greatly facilitated when one chooses such conditions for the reverse transcriptase reaction that not only favors the synthesis of full-length sequences, but also suppress the synthesis of small DNA chains. Preferred reaction conditions for reverse transcriptase from avian myeloblastosis virus are given in the examples section. The specific parameters that can be varied to achieve maximum production of long chain, high fidelity DNA translations are the reaction temperature, salt concentration, enzyme level, primer concentration relative to the template, and reaction time.

The conditions of temperature and salt concentration are chosen to optimize the specific base pairing between the oligo dT primer and the polyadenylated portion of the RNA template. Under suitably chosen conditions, the primer is capable of binding to the polyadenylated region of the RNA template, whereby nonspecific initiation due to binding at other sites on the template, such as short A-rich sequences, is substantially prevented. The effects of temperature and salt concentration are mutually dependent. Higher temperatures and lower salt concentrations reduce the safety of specific base-pairing effects. The reaction time is kept as short as possible to be non-specific

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To prevent initiations and to minimize the opportunity of mining. Reaction times are temperature dependent, with lower temperatures requiring longer reaction times. At 42 ° C, reaction times of 1 to 10 minutes are suitable. The primer should be present in 50 to 50 fold molar excess over the RNA template and also the enzyme should be present in similar molar excess over the RNA template. The use of excess enzyme and primer promotes initiation and chain growth of the cDNA so that long-chain cDNA transcripts are obtained within the short incubation times.

In many cases it will be possible to carry out the remainder of the purification process according to the invention with single-stranded cDNA sequences obtained by transcription from mRNA. However, as will be described below, there are cases in which the desired restriction enzyme acts only on double-stranded DNA. In these cases, the cDNA generated in the above manner can be used as a template for synthesis of the double-stranded DNA using a DNA polymerase such as reverse transcriptase and a nuclease capable of hydrolyzing single-stranded DNA. Methods for producing double-stranded DNA in this manner have already been described, see, for example, Ullrich, A., Shine, J., Chirgwin, J., Pictet, R., Tischer, E., Rutter, WJ, and Goodman, HM, Science 196, 1313 (1977).

Heterogeneous cDNA obtained by transposing heterogeneous mRNA sequences is then treated with one or two restriction endonucleases. The choice of endonuclease depends primarily on a preliminary test that identifies the recognition sites for the enzyme in the sequence of the cDNA to be isolated. The method is based on the presence of two such sites. If these are identical, then a single enzyme is sufficient. The desired sequence is generated by cleavage at two sites, eliminating size differences in the desired cDNA sequence, and yielding a number of molecules - called fragments - which have the desired sequence and are homogeneous in length. If the restriction sites are different, two enzymes are needed to generate the fragments of homogeneous length.

The choice of restriction enzyme (s) capable of producing a nucleotide sequence fragment of optimal length which encodes all or part of the desired protein must be empirical. If the amino acid sequence of the desired protein is known, one can compare the nucleotide sequence of nucleotide fragments of uniform length, which has arisen by cleavage with restriction endonucleases, with their coding amino acid sequence, based on the known relationship of the genetic code. However, a complete amino acid sequence of the desired protein is not required since a sufficiently accurate identification based on a partial sequence can be made. If the amino acid sequence of the desired protein is unknown, the polynucleotides of equal length generated by cleavage with the restriction endonuclease can be used as assay reagents capable of identifying the synthesis of the desired protein in a suitable in vitro protein synthesis system. The mRNA can also be purified by affinity chromatography or other techniques that appear appropriate to one skilled in the art.

The number of suitable restriction enzymes depends on whether single-stranded or double-stranded cDNA is used. Preferred are enzymes capable of acting on single-stranded DNA, since the latter is the direct reaction product of mRNA transcription. The number of restriction enzymes of which the ability to act on single-stranded DNA is known is limited. At present, the enzymes Haelll, Hhal and Hin (f) I appear to be suitable. The enzyme MboII also acts on single-stranded DNA. As further studies reveal the effect of other restriction enzymes on single-stranded DNA, these enzymes can easily be included in the list of preferred enzymes. In addition, enzymes that act on double-stranded cDNA are suitable. However, they are not preferred because additional reactions are required to produce the double-stranded cDNA that offer opportunities for loss of long sequences and other losses in work-up. Working with double-stranded cDNA has the further technical disadvantage that the subsequent sequence analysis is more complicated and cumbersome. For these reasons, the single-stranded cDNA is preferred, but the use of double-stranded DNA is possible.

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The cDNA prepared for treatment with the restriction endonuclease can be radiolabeled so that it can be detected by later separation techniques. A preferred method is the incorporation of a radioactive label, such as³²P in α-position of one of the 4 deoxynucleoside triphosphates. The highest activity is achieved when the concentration of the radioactive precursor is high in relation to the concentration of the non-radioactive form. However, the total concentration of each deoxynucleoside triphosphate should be greater than 20 μΜ in order to maximize the length of cDNA obtained in the reverse transcriptase reaction; see. Efstratiadis, A., Maniatis, T., Kafatos, FC, Jeffrey, A., and Vournakis, JN, Gell. 4, 367 (1975). For the determination of the nucleotide sequence of the cDNA, the 5 'ends with³²P be marked, u.zw. in a reaction catalyzed by the enzyme polynucleotide kinase, cf. Maxam, AM, and Gilbert, W., Proc. Nat. Acad. Sci. USA, 74, 560 (1977).

Fragments generated by the action of a restriction enzyme or two restriction enzymes may be separated from each other and heterodisperse sequences having no recognition sites by any suitable technique capable of separating polynucleotides due to differences in length. These methods include the various electrophoretic methods and sedimentation methods in the ultracentrifuge. Gel electrophoresis is preferred because it gives the best separation in terms of polynucleotide length. Besides, this method allows quantitative isolation of separate materials. Methods for gel electrophoresis have been described by Dingman, CW, and Peacock, AC, Biochemistry T_, 659 (1968), and Maniatis, T., Jeffrey, A., and van de Sande, H., Biochemistry 14, 3787 (1975) ,

Without treatment with restriction endonuclease, the cDNA transcripts obtained from most sources are heterodisperse in length. By the action of a suitably chosen restriction endonuclease or a pair of such endonucleases, the polynucleotide chains containing the desired sequence are cleaved at the restriction sites so that raan receives polynucleotide fragments of equal length. After gel electrophoresis, they form a distinct band. Depending on the presence or absence of restriction sites on other sequences, other discrete bands can arise, but most likely to correspond to a different length than that of the desired sequence. As a result of the action of the restriction endonuclease, the result of gel electrophoresis is the appearance of one or more discrete bands while the remainder of the cDNA remains heterodisperse. If the desired cDNA sequence comprises the majority of the polynucleotide present, the result of electrophoresis shows that most of the cDNA is in the discrete band.

Although it is unlikely that two distinct sequences will result in fragments of virtually equal length upon restriction enzyme cleavage, one method of determining the purity of the fragments of defined length would be desirable. The sequence analysis can be used to detect impurities that make up 10% or more of the total material of the band in question. As part of the invention, a method for finding smaller amounts of impurities has been developed based on the same principles as the isolation method described above. The method requires that the nucleotide sequence fragment have a recognition site for a restriction endonuclease that was not used in the previous isolation. Treatment of a polynucleotide material eluted from a gel electrophoresis band with a restriction endonuclease capable of interacting internally with the subject sequence will result in cleavage of this sequence into two subfragments of probably different lengths. In electrophoresis, these subfragments form two discrete bands at the sites corresponding to their lengths, the sum of which is equal to the length of the polynucleotide before cleavage. Impurities in the original band that are not attacked by the restriction enzyme should migrate to their original position. Impurities that provide one or more recognition sites for the enzyme should result in two or more subfragments. Since it is believed that the distribution of recognition sites is essentially random, the likelihood that an impurity gives subfragments of the same size as the fragment of the desired sequence is extremely small. The amount of radioactively-labeled polynucleotide in each band can be quantified by measuring the radioactivity present in each band or by another suitable method. A quantitative one

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Measurement of the purity of the fragments of the desired sequence can be obtained by measuring the amounts of substances corresponding to the subfragments from the desired sequence with the whole

Substance quantity compares.

After the separation has taken place, the desired sequence can then be restored. For this purpose one can use the enzyme DNA ligase, which catalyses the end-to-end linkage of DNA fragments. The bands of gel electrophoresis corresponding to the subfragments of the desired sequence can be separately eluted and then combined in the presence of DNA ligase under the appropriate conditions, cf. Sgaramella, V., Van de Sande, JH, and Khorana, HG, Proc. Nat. Acad. Sci. USA, 67, 1468 (1970). If the sequences to be joined are not blunt-ended, one can use ligase from E. coli, cf. Modrich, P., and Lehman, IR, J. Biol. Chem. 245, 3626 (1970).

Restoration of the original sequence from subfragments obtained by treatment with restriction endonuclease is greatly enhanced by the use of a method that prevents recovery in the wrong sequence. This undesirable result is prevented by treating the cDNA sequence of desired sequence and homogeneous length prior to cleavage of the homogeneous cDNA with a restriction endonuclease with a reagent capable of removing the 5'-terminal phosphate groups. As the enzyme, alkaline phosphatase is preferred. The 5'-terminal phosphate groups are a structural prerequisite for subsequent linkage by DNA ligase, which is used to restore the subfragments. Thus, ends lacking a 5'-terminal phosphate group can not be covalently bound. The DNA subfragments can only be linked at the ends which have a 5 'phosphate group, these being generated by the cleavage of the isolated DNA fragments with the restriction endonuclease. The method is described in detail in DE-OS 2822568.

The majority of the cDNA transcripts, under the conditions used, are from the mRNA region comprising the 5 'end of the mRNA template by specific primer action on this template by a fragment obtained by restriction endonuclease cleavage. In this way, the method described above can be used to generate not only fragments having a specific nucleotide sequence with respect to a desired protein, but also the entire nucleotide sequence encoding the protein of interest.

The purification process is of particular importance in the cloning of human genes, which can be spent under the US regulations only after very thorough purification in recombinant DNA and then in bacteria, or if working in accordance with secure (P4) facilities, see. US Federal Register, Vol.41, No.131.7.7.1967, p.27902 to 27943. The present method allows the production of sufficiently pure human genes that have the essential structure of HCS and HGH. Human gene material which has been isolated and purified in the manner described above can be introduced into recombinant plasmids or other transfer vectors. Double-stranded chemically synthesized oligonucleotide linkers having the restriction endonuclease recognition sequence can be attached to the ends of the isolated cDNA to facilitate subsequent enzymatic removal of the gene portion from the transfer vector DNA. Scheller, RH, et al, Science 196, 177 (1977). The DNA of the transfer vector is converted from a closed loop to a linear form by treatment with a corresponding restriction endonuclease. The resulting ends are treated with alkaline phosphatase to remove the 5'-phosphate groups so that the DNA of the transfer vector does not form a continuous loop again upon reaction with DNA ligase before first inserting a segment of human DNA , The cDNA with the oligonucleotide linker and pretreated acceptor vector DNA are mixed with a DNA ligase to link the cDNA to the vector DNA to form a closed loop of recombinant vector DNA with incorporated cDNA. When a plasmid is used as the transfer vector, the closed loop is usually the only one capable of transforming a bacterium. Transformation is the process by which a microorganism incorporates extracellular DNA into its own genetic makeup. Closed loop plasmid DNA can be incorporated under appropriate environmental conditions. The closed loop plasmid is replicated in the transformed cell and the replicated copies are distributed to cell progeny on cell division. The result is a new cell strain that contains the plasmid and carries its genetic determinants. Such transformation by a plasmid while retaining the plasmid genes in plasmid replication occurs with great frequency when the transforming plasmid DNA is in the form of a closed loop, but not at all or rarely when linear plasmid DNA is used. Once a recombinant transfer vector is obtained, transformation of a suitable microorganism is a straightforward process, and novel microorganism strains containing the human gene can be readily isolated using the appropriate selection methods known per se.

Using the procedures for purification and analysis described above, a nucleotide sequence was isolated which contains the major portion of the structural gene for HCS, the purity of which proved greater than 99%. The structural gene for HGH was isolated in a comparable degree of purity. New plasmids containing the isolated HCS or HGH sequences were synthesized. Furthermore, new microorganisms have been produced which contain the isolated HCS or HGH sequences as part of their genetic material.

The drawings demonstrate the results achieved in the examples.

Fig.l is an autoradiogram of a series of gel electrophoresis experiments with ³²P-labeled cDNA (see Example 1).

Fig. 2 is the schematic representation of the HCS-encoding nucleotide sequence showing the relative position of various restriction sites (see Example 1).

Fig. 3 is an autoradiogram of gel electrophoresis experiments ^3sP-labeled cDNA (see Example 2).

Figures 4 and 5 are autoradiograms of gel electrophoresis experiments with ³²P-labeled cDNA (see Example 3).

Example 1:

The general procedure for isolating a specific cDNA sequence is demonstrated by isolating a sequence comprising part of the coding region for HCS and extracted from placental tissue.

Extraction of the mRNA from the placenta

Human birth placentas, obtained in cesarean delivery, were rapidly frozen in liquid nitrogen and stored at -60 ° C. For extraction of whole RNA, 40 g of frozen placental tissue was broken into small pieces and mixed in 140 ml of a freshly prepared solution of 7 M-guanidinium HCl (see Cox, RA, Methods in Enzymology 12, 120 [1968]). , 20mM Tris-HCl, p_H 7.5, 1 mM EDTA, 1% sarcosyl (trade name of Ciba-Geigy Corp., Greenshoro, NC) dissolved at 0 ° C. After addition of 0.5 g of cesium chloride per ml, the dark brown solution was heated to 65 ° C for 5 min, cooled rapidly in ice, onto a pad of 5.7 M CsCl, 10 mM Tris-HCl, p_H = 7.5, 1 M EDTA in 2.5 x 8.75 cm nitrocellulose tubes and centrifuged in an SW27 rotor (Beckman Instruments Corp., Fullerton, California) at 27,000 rpm for 16 hours at 15 ° C (Glisin, V., Crkvenjakov, R., and Ryus, C., Biochem., 13, 2633 [1974]). After centrifuging, it was decanted, the tubes were dehydrated, and the 1/2 cm bottom piece containing the pure RNA pellet was cut with a razor. The pellets were transferred to a sterile Erlenmeyer flask and suspended in 20 ml of 10 mM Tris HCl, p_H = 7.5, 1 mM EDTA, 5% sarcosyl and 5% phenol. The solution was then made 0.1 molar of sodium chloride and shaken vigorously with 40 ml of a mixture of 50% phenol and 50% chloroform. The RNA was precipitated from the aqueous phase with ethanol in the presence of 0.2 M Na-acetate, p * j = 5.5. The RNA pellets were washed with 95% ethanol, dried and dissolved in sterile water. Usually, 40 g of placental tissue gives about 30 mg of RNA, after which about 300 pg of polyadenylated RNA is obtained after twice chromatographing on oligo-dT-cellulose, cf. Aviv and leather, loc.cit.

Synthesis of the cDNA

Analytical reactions were carried out in 5 μl containing 50 mM Tris-HCl, Pjj = 8.3, 0.1 mM EDTA, 7 mM MgCl, 20 mM KCl, 10 mM β-mercaptoethanol, 40 μM dCTP (50,000 cpm P / p mol ), 500 pM dGTP, dATP and dTTP, 100 pg / ml polyadenylated RNA, 20 pg / ml oligo-dT₁₂_i_8th (to acquire

14 No. 369386 to Collaborative Research, Waltham, Mass.) And 100 units / ml of reverse myeloblastosis virus reverse transcriptase. The enzyme may be obtained from Life Science Intercorporated, St. Petersburg, Florida, which has been contracted with the National Institutes of Health by the method of Kacian, DL, and Spiegelman, S., Methods in Enzymology 29, L.Grossman, and K.Moldave (eds) Academic Press, NY (1974), p.150. The reactions were initiated by addition of the enzyme at 0 ° C, the synthesis was carried out at 42 ° C for 6 min. Under these conditions, 10® cpm³²P was incorporated into the trichloroacetic acid precipitable material, and each pg RNA gave about 50 ng of cDNA. In order to obtain enough cDNA for a sequence analysis, the reaction volumes were increased to 100 pl and the dCTP concentrations to 250 pM (specific activity 500 cmp^3!P / mol). Under these conditions were about 200,000 cpm^3sP-labeled dCTP introduced into cDNA.

Treatment with restriction endonuclease

For digestion with restriction endonuclease, the analytical reactions were stopped by addition of 20 μl of ice-cold water, then boiled for 2 minutes, rapidly cooled on ice and made 7 mM with magnesium chloride. Samples of 5 pl each (2 x 10⁵ cpm) were digested using an excess of the restriction endonuclease (s) Haell I or Hhal, or both, at 37 ° C for 1 h. The enzyme Haelll was by the method of Middleton, JH, Edgell, MH, and Hutchinson, GA III, J. Virol. 10, 42 (1972). The enzymes Hhal and Hpall had been obtained from the New England Bio-Labs, Beverly, Mass. Haelll is also available from this manufacturer. The amount of enzyme was determined empirically to be in excess of the amount necessary to completely digest an equivalent amount of restriction-sensitive DNA under identical reaction conditions. The reactions were quenched with 5 μl of 20 mM EDTA, 20% sucrose, 0.05% bromophenol blue, heated to 100 ° C for 1 min and then analyzed by polyacrylamide gel electrophoresis.

Separation of the products was carried out with a 4.5% -10% polyacrylamide slab gel during 2 1/2 h at 150 V in Tris-borate-EDTA buffer (Fingman, CW, and Peacock, AC, loc. Cit.). the visualization was done by autoradiography of dry gel.

Fig.l shows the results of electrophoresis and autoradiography of ³²P-labeled cDNA, the preparation of which is described above. The samples migrated electrophoretically through 4.5% acrylamide and then through 10% acrylamide. On the left, a line indicates the boundary between the two gel areas. Band A represents the electrophoretic migration of the entire cDNA transcript, Band B shows the migration of the H-treated cDNA. Volume C shows the migration of Haelll-treated cDNA. Band D shows the electrophoretic migration of the whole cDNA treated with both Hhal and Haelll. Band E shows the electrophoretic migration of the material isolated from the major band Band C. Band F shows the electrophoretic migration of material isolated from the major band of band C after treatment with Hhal. Band G shows the electrophoretic migration of a 5'- split with Haelll³²P end-labeled single-stranded phage M13 DNA, which was used as a size standard, s. Horiuchi, K., and Zinder, ND, Proc.Nat.Acad.Sci. USA, 72, 2555 (1975). The approximate lengths of these DNA fragments in nucleotide units are indicated by the numbers to the right.

The result in band A shows that the cRNA transcript from mRNA birth placenta is heterodisperse. Treatment with Hhal (Band B) or Haelll (Band C) results in the accumulation of polynucleotides of discrete length. The preparation of such discrete bands indicates that in a heterogeneous population of cDNA transcripts there is at least one multiple copy sequence having two Hhal and Haelll restriction sites, respectively. Cleavage with Hhal produces a fragment of about 470 nucleotides, and Haelll gives a fragment of about 550 nucleotides in length. Digestion with both enzymes results in three fragments, namely A 90 nucleotides in length, B 460 nucleotides in length, and C about 10 nucleotides in length. On

Due to its small size, fragment C migrates out of the gel under the conditions used in FIG. The band appearing at the interface between 10 and 4.5% gel is a heterogeneous material which was too large to enter the 10% gel and therefore accumulated at the interface. As can be concluded from the simple banding pattern of band D, fragments A and B appear to be from the same cDNA molecule. This conclusion is made by the

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Elution of the larger Haelll fragment confirmed from the gel, which is again digested with Hhal. This treatment yields two fragments that migrate in the same way as the bands that result from combined digestion of the entire cDNA with Haelll and Hhal, as can be seen by comparing bands D and F. In the digestion product of the entire cDNA according to Volume D, the autoradiographic density, which is a measure of the total radioactivity present, is greater for fragment A than for fragment B, although the opposite might be expected due to the size differences. This observation suggests that fragment A is formed by transcription of a region closer to the 3 'end of the mRNA as fragment B.

Figure 2 is a schematic representation of the cDNA molecule with the relative placements of the Haelll and Hhal restriction sites. DNA fragments A and B, derived from the same cDNA molecule, were located in the autoradiogram shown in Figure 1, panel D, for their relative intensity. The existence of the DNA fragment C was inferred from the difference in electrophoretic mobility of the bands appearing in bands B and D of Fig. 1. The size of DNA fragment A is well known due to a determination of the nucleotide sequence by the method of Maxam, A., and Gilbert, W., loc. Cit. The size of DNA fragment B was determined by comparison with M13 DNA size markers according to FIG.

The nucleotide sequence of DNA fragment A and part of the 5 'end of fragment B were performed according to the method of Maxam, A., and Gilbert, W., loc. Cit. Since the amino acid sequence of HCS is known, the nucleotide sequence of the two fragments can be compared to the amino acid sequence using the known relationship of the genetic code. Based on this relationship, it could be demonstrated that the specific sequences actually encode parts of the HCS molecule, and furthermore the arrangement of these fragments according to FIG. 2 could be confirmed.

Example 2:

The following example demonstrates the ability of the method of the invention to purify a nucleotide sequence present as a minor proportion in the entire population of nucleotide sequences. Defined RNA mixtures containing purified rabbit globin RNA and human polyadenylated placental RNA were used as template, the reaction with reverse transcriptase being in the presence of³²P dCTP with a specific end activity of 10⁵ cpm / pmol. The cDNA products were cleaved with the endonuclease Haelll and the cleavage products were separated on 4.5% -10% polyacrylamide slab gel. The cDNA fragments were visualized by autoradiography of the dried gel.

3 shows the results of the experiments. The gel electrophoresis was carried out essentially as described in Example 1. Bands A and H denote the size markers obtained by cleavage of phage M13 DNA with the endonuclease Haelll and 5'-³²P-end labeling of the resulting fragments. The approximate lengths of these DNA fragments in nucleotides are indicated by the numbers on the left. Bands B to G show the electrophoresis patterns obtained after carrying out the above reaction sequence on mixtures of globin RNA and placental RNA with changing the amounts, as shown in the following table:

<td>tape</td><td>Globin RNA ng</td><td>Pacenta RNA ng</td>

<td>B</td><td>300</td><td>0</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>285</td>

<td>F</td><td>7.5</td><td>292.5</td>

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

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It can be seen that the globin cDNA gives a Haelll fragment of 320 nucleotides in length. The

Globin cDNA transcript can still be detected if the globin RNA is only 2 to 5% of the total

RNA. If an RNA species after isolation is too low to carry out this analysis, it may be partially purified by one of the known RNA purification techniques until it is about 2 to 5% of the remainder of the mixture.

Example 3:

This example describes the purification of a nucleotide sequence fragment of about 550 base pairs in length which contains part of the coding region for HCS and a method for measuring the purity of the isolated sequence. The purity of the purified fragment was more than 99%.

Purification of HCS cDNA

Polyadenylated placental RNA isolated according to the protocol of Example 1 was enriched in HCS mRNA by sedimentation in a 5 to 20% (w / v) sucrose gradient of 4 ° C in the SW 27 rotor Beckman ultracentrifuge at 25,000 rpm for 16 h. The ILS 14S region of the gradient was collected and 100 pg of this RNA was used to synthesize the double-stranded cDNA according to the protocol of Ullrich, A., et al., Loc. Cit. The synthesis of the second strand was stopped by extraction of the reaction mixture with 1 part by volume of ethanol at -70 ° C. The digestion of the Haell endonuclease cDNA was performed in 50 μl of 6 mM Tris-HCl, Pr = 7.5, 6 mM MgCl₂ , 6mM β-mercaptoethanol with 2 units of HaCl at 37 ° C for 2 hours, followed by treatment with 0.1 units of bacterial alkaline phosphatase (BAPF type, Worthington Biochemical Corp., Freehold, NJ, definition of units by the manufacturer) 60 ° C for 10 min. After extraction with 1 part by volume of phenol / chloroform, the DNA was precipitated with 2 parts by volume of ethanol at -70 ° C, dissolved in 20 μl of 10 mM Tris-HCl, pH = 8, 1 mM EDTA and electrophoresed at 6% (w / v) polyacrylamide gel. Fig. 4 (F) shows the electrophoresis pattern of this reaction mixture showing a prominent band corresponding to a nucleotide sequence of about 550 base pairs in length. The 550 base pair fragment was excised from the gel and eluted electrophoretically, the result being shown in Fig. 4 (E).

The remaining material corresponding to the 550 base pair fragment of Figure 4 (E) was treated with 4 units of Hhal endonuclease in 50 μl of the same buffer used for HaellI endonuclease digestion for 2 hours at 37 ° C. After extraction with phenol / chloroform and precipitation with ethanol, the products of digestion were separated by electrophoresis on a 6% (w / v) polyacrylamide gel. The result is shown in Fig. 4 (D).

The two fragments were eluted electrophoretically, pooled and re-linked by incubating for two hours at 15 ° C in 20 μl of 66 mM Tris-HCl, pI = 7.6, 6 mM MgCl₂, 15 dM dithiothreitol, 1 mM ATP containing 20 pg / ml T4 DNA ligase. The reaction mixture was then diluted to 200 μl with 0.1 molar sodium chloride solution and extracted with 1 part by volume of phenol / chloroform, the DNA was precipitated with 2 parts by volume of ethanol. After resuspension in 20 μl of 10 mM Tris-HCl, p_H 8, 1 mM EDTA, the linkage products were separated by electrophoresis on 6% (w / v) polyacrylamide gel. The result is shown in Fig. 4 (C). From the electrophoresis pattern of Fig. 4 (C), it can be seen that the 550-nucleotide fragment has been restored by the linking treatment. Preliminary treatment with alkaline phosphatase ensured that the two Hhal fragments were linked in the original order. The further bands according to FIG. 4 (C) correspond to the dimer formation between Hhal fragments, since this is not prevented by the treatment with alkaline phosphatase.

The 550 nucleotide fragment was excised from the gel and eluted electrophoretically. The electrophoresis pattern of this material is shown in Fig. 4 (B). Fig. 4 (A) is the electrophoresis pattern of a³³P-labeled Haelll digestion product of double-stranded M13 DNA used for size labeling. Electrophoretic analyzes were performed in 6% (w / v) polyacrylamide gel in 5 mM tris-borate, p_H = 8, 1 mM EDTA at 100 V for 2 h. After electrophoresis, the gel was dried and autoradiograms were made using a Kodak NS2T x X-ray film.

Purity of the recovered 550 nucleotide fragment of HCS cDNA

The isolated, recovered HCS cDNA HaeIII fragments were at the 5 'end with ³²P labeled using the enzyme polynucleotide kinase from infected with bacteriophage T4

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E.coli, s. Panet, A., et al., Biochemistry 22, 5045 (1973). Polynucleotide kinase is available from PL-Biochemical, Milwaukee, Wisconsin. The fragment was then digested with either Hhal or Hpall in 50 μl of 6 mM Tris-HCl, p_H = 7.6, 6 mM MgCl_a , 6 mM β-mercaptoethanol at 37 ° C for 2 h digested. After extraction with an equal volume of phenol / chloroform, the DNA was precipitated with 2 volumes of ethanol at -70 ° C, resuspended in 20 μl, 10 mM Tris-HCl, p = 8, 1 mM EDTA and electrophoresed. With an X-ray film, the labeled fragments were visualized as described above.

The results are shown in FIG. Figure 5 (B) and 5 (E) show repeats with the 550 nucleotide fragment prior to restriction enzyme treatment. Fig. 5 (C) shows the pattern after Hhal splitting and Fig. 5 '(D) after Hpall splitting.

The purity of the 550 nucleotide fragment was measured by dissecting the autoradiogram of the cleavage products resulting from the restriction enzyme and quantifying the distribution of radioactivity in both digestion products. These measurements show that the reconstituted Haelll fragment of human HCS cDNA was more than 99% homogeneous.

Example 4:

This example describes the synthesis of a plasmid containing a nucleotide sequence of 550 base pairs, which constitutes the major portion of the coding region for HCS.

Following the protocol of Example 3, a 550 nucleotide fragment of HCS cDNA greater than 99% pure is prepared. The terminal 5'-phosphate groups are reconstituted in a reaction mixture containing 50mM Tris-HCl, p ^ = 8.5, 10mM MgCla, 0.1mM spermidine, 5mM β-mercaptoethanol, 5% (w / v. ) Glycerol, 333 pmol ATP, 5 units T4 polynucleotide kinase, incubated for 2 hours in a final volume of 40 μl at 37 ° C. The DNA is isolated from the reaction mixture by phenol extraction and precipitation with ethanol. Then, synthetic decanucleotide linkers having a restriction specificity for EcoRI having the sequence 5'-CCGAATTCGG-3 '(produced by Scheller, et al., Loc.cit.) Are attached to the HCS DNA in a molar ratio of about 50: 1 in 50 μl of 66 mM Tris-HCl, p_H = 7.6, 9 mM MgCl_a , 15 mM dithiothreitol, 1 mM ATP and 20 pg / ml T4 DNA ligase. The links are available from Collaborative Research, Waltham, Massachusetts. After 18 hours of incubation at 4 ° C, the reaction is stopped by extraction with phenol / chloroform. The linkage products are precipitated with ethanol, redissolved in 50 μl of 100 mM NaCl, 50 mM Tris-HCl, pK = 7.6, 7 mM MgCl_a and digested with 50 units of EcoRI endonuclease at 37 ° C for 2 hours. Digestion with the endonuclease results in cleavage at the EcoRI site of the decamers to yield HCS cDNA with EcoRI cohesive ends as well as cleaved unreacted decanucleotides and self-linked decanucleotides. Since the cleaved decamers also have EcoRI end groups and would compete with the HCS cDNA when recombined with the similarly cleaved plasmid, the HCS cDNA is isolated by gel electrophoresis prior to reaction with the transfer vector. The use of the above decanucleotide linkers has the advantage that the HCS cDNA fragment can be isolated from the plasmid in a form identical to the original fragment.

The transfer vector used was the bacterial plasmid pMB-9, a molecule of molecular weight 3.5 × 10⁶having a single Eco RI site (manufactured by Rodriguez, RL, Bolivar, F., Goodman, HM, Boyer, HW, and Betlach, M. ICN-UCLA Symposium On Molecular and Genetic Biology, DP, Witra, WJ Rutter, and CFFox ( Ed.), Academic Press, New York [1976], pp. 471-477). Plasmids pMB-9 and pBR-322 (see Example 5) are available from Bethesda Research Labs, Rockville, Maryland. Infection of E. coli with pMB-9 confers resistance to tetracycline. The incorporation of DNA into the EcoRI position of pMB-9 does not alter the tetracycline resistance nor any other known property of the plasmid. Therefore, there are no phenotypic differences between recombinant and normal plasmids. The EcoRI digested pMB-9 was thus first treated with alkaline phosphatase according to the method of DE-OS 2822568, cf. also Ullrich et al., loc. cit. Treatment with alkaline phosphatase removes the 5'-phosphate groups from the EcoRI-generated ends of the plasmid and prevents self-assembly of the plasmid DNA, thereby ensuring that ring formation and later transformation become dependent upon the insertion of a DNA fragment with S'- phosphorylated ends. The treatment with alkaline phosphatase was carried out in a reaction mixture with 1.0

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Enzyme units per mg of plasmid DNA in 25 mM Tris-HCl, ρθ = 8 for 30 min at 65 ° C. Phenol extraction was then performed to remove the phosphatase and precipitate the DNA with ethanol. The linkage of the HCS cDNA with the pMB-9 thus treated was carried out in 50 μl reactions containing 60 mM Tris-HCl, pI = 8, 10 mM β-mercaptoethanol, 9 mM MgCl 2, 10 to 50 ng of the purified HCS cDNA and about 500 ng EcoRI digested 5'-dephosphorylated plasmid DNA. The reactions were initiated by addition of T4 DNA ligase to 5 μg / ml, run for 1 h at 15 ° C and then the mixture was diluted to 0.25 ml with 120 mM NaCl, 1 mM EDTA. The diluted reaction mixture was used directly to transform E. coli X-1776.

E.coli X-1776 is a host organism specifically designed for recombinant DNA technology, referred to as the EK-2 host by the National Institute of Health. The strain can be obtained from Dr. Red Curtiss III, University of Alabama, Department of Microbiology, Birmingham, Alabama. Bacteria are grown in 150 ml nutrient broth supplemented with 100 pg / ml diaminopimelic acid (DAP) and 40 pg / ml thymine, to a cell density of approximately 2 -10^8th Cells / ml. The cells are collected by centrifugation and washed in 60 ml of 10 mM NaCl, recentrifuged and suspended in 60 ml of transformation buffer containing 10 mM Tris-HCl, p_H = 8, 140 mM NaCl, 75 mM CaCl₂ contains. The cell suspension is kept on ice for 15 min, then the cells are spun down and suspended in 1.5 ml of the same transformation buffer. 0.5 ml of the cell suspension is added to 0.5 ml of diluted coupling reaction mixture, followed by incubation on ice for 15 min. After 4 min at 25 ° C, the mixture is again kept on ice for 30 min. 0.2 ml of the cell suspension is transferred directly to nutrient agar plates supplemented with 100 pg / ml DAP, 40 pg / ml thymine and 20 pg / ml tetracycline. Four transform samples are obtained, each containing a 550 base pair insert released from the plasmid DNA by digestion with EcoRI or Haelll endonuclease.

For sequence analysis, a transformation clone designated pHCS-1 was selected. E. coli X-1776 (pHCS-1) was grown in a suitable nutrient medium, the plasmid DNA was isolated therefrom and cleaved with EcoRI endonuclease. The 550 base pair insert was isolated from the linear pMB-9 by electrophoresis in 6% polyacrylamide gel followed by DNA sequence analysis according to the method of Maxam and Gilbert, loc. Cit. Subfragments of HCSDNS were prepared by incubation with Hpall restriction endonuclease and the 5 'ends were probed using γ³³Ρ-ΑΤΡ and polynucleotide kinase labeled. After sequence analysis according to Maxam and Gilbert, the nucleotide sequence of the cloned HCS DNA was determined. By comparison with the known amino acid sequence of the HCS, it was found that the 557 nucleotide sequence represented that part of the HCS mRNA coding region from amino acids 24 to 191 plus 50 nucleotides of the 3 'untranslated region, cf. Niall, HD, Hogan, ML, Sauer, R., Rosenblum, IY, and Greenwood, FC, Proc.Nat.Acad.Sci. USA, 68, 866 (1971). The primary structure of the HCSmRNA, determined from the DNA sequence of the cloned fragment pHCS-1, is shown in Table 3, together with the resulting amino acid sequence corresponding to the genetic code. The amino acid sequence resulting from the nucleotide sequence is identical to the recently published chemically determined amino acid sequence. This shows that the initially isolated HCSmRNA was copied with great accuracy in vitro, and that the cloned HCS DNA fragment was replicated in the transformed bacterium with great accuracy.

Table 3

Nucleotide sequence of a strand of HCS DNA from cloned pHCS-1

The numbers refer to the amino acid sequence beginning at the amino terminus. The DNA sequence shown corresponds to the mRNA sequence for HCS, except that in the mRNA T is replaced by U. Further, the amino acid sequence of positions 1 to 23 is shown.

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Example 5:

This example describes the purification of DNA whose nucleotide sequence comprises most of the coding region for HGH and the synthesis of a plasmidic transfer vector containing the purified DNA. Also described is the production of a strain of microorganism containing the DNA as part of its genetic makeup. The HGH had been purified for HCS essentially according to the procedure of Example 3, except for the following changes.

Human benign pituitary tumors frozen in liquid nitrogen after surgical removal and each weighing 0.4 to 1.5 g were suspended in 4 molar guanidinium thiocyanate solution buffered at mercaptoethanol in molar ratio and at p = 5.0. thawed at 4 ° C and homogenized. The homogenate was over 1.2 ml of 5.7 molar cesium chloride solution ,. which contained 100 mmol of ethylenediaminetetraacetic acid, layered and centrifuged at 37 rpm in the SW 50.1 rotor of a Beckman ultracentrifuge at 15 ° C. for 18 h. The RNA came to the bottom of the centrifuge tube. The further purification in a column with OligodT and sucrose gradient sedimentation was carried out as described in Examples 1 and 3. About 10% of the thus isolated RNA-encoded growth hormone was estimated to be from the incorporation of a radioactive amino acid precursor in anti-growth hormone precipitate in a cell-free wheat germ cell translation system; see. Roberts, BE, and Patterson, BM, Proc.Nat.Acad.Sci. USA, 70, 2330 (1973). Single-stranded and double-stranded cDNAs were prepared as described in Example 3. The HGH cDNA was then treated with the restriction endonuclease Haelll and alkaline phosphatase as described in Example 3, followed by fractionation by gel electrophoresis. A discrete band of approximately 550 nucleotides in length was observed and isolated for further purification.

For further purification, the above-described technique of subdividing DNA into subfragments, purifying these subfragments separately, and recombining them was performed, but in the case of HGH, the restriction endonuclease PvuII was used, containing two subfragments of about 490 and about 60 nucleotides in length, respectively revealed. All restriction enzymes used are commercial products of New England Biolabs, Beverly, Massachusetts. The newly linked product of about 550 base pairs in length was more than 99% pure, as seen by subfractionation in 4 different restriction endonuclease systems.

The synthesis of a recombinant transfer vector containing HGH DNA was carried out essentially according to the procedure of Example 4, but differing the decanucleotide linker and plasmid. A decanucleotide linker with Hind IXI specificity was used, containing the sequence 5'-CCAAGCTTGG-3¹ possessed. Treatment with Hsul yielded cohesive-ended HGH cDNA. Hsul and Hind III have the same cleavage site specificity and can be used in exchange. The transfer vector used was plasmid pBR-322. It gives the host resistance to the antibiotics ampicillin and tetracycline. DNA insertion into the Hind III site reduces or eliminates tetracycline resistance. The selection of recombination was therefore due to growth on ampicillin-containing nutrient plates and due to the inability to grow to 20 pg / ml tetracycline. The HGH cDNA was recombined with Hsul-digested and alkaline phosphatase-treated pBE-322 using essentially the conditions of Example 4.

The products of the ligase reaction were used to transform E. coli X-1776 under the conditions of Example 4. Seven colonies were isolated for their ability to grow in the presence of ampicillin and their inability to grow in the presence of tetracycline. Five of the seven colonies contained the recombinant plasmid, which had the portion of the HGH DNA of about 550 base pairs. One of the bacterial strains, pHGH-1, carrying the HGH DNA as part of its genetic makeup was grown in sufficient quantity to form a source of plasmid DNA from which the HGH DNA was treated by treatment with Hind III or Hsul could be reisolated. This isolated HGH DNA, which had undergone numerous replications, was subjected to the sequence analysis according to Example 4, with the following result:

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Table 4

Nucleotide sequence of a strand of HGH DNA from cloned pHGH-1

The numbers refer to the amino acid sequence of HGH beginning at the amino terminus.

The DNA sequence shown is in agreement with the mRNA sequence for HGH, except that in the mRNA T is replaced by U.

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The invention provides for the first time a generally applicable method for purifying specific nucleotide sequences. These sequences can be related to the production of a particular protein of technical or medical importance. The process results in purified nucleotide sequences, which may be fragments of larger sequences encoding the desired protein. The method of the present invention can be used in combination with known ancillary methods to produce the entire nucleotide sequence encoding a particular protein.

The invention also allows the high purification of a nucleotide sequence of specific length and of any origin. Further, a method of measuring the degree of purity of such fragments is disclosed. In accordance with the present invention, a nucleotide sequence encoding a portion of human HCS having a purity of at least 99% was isolated.

Transfer vectors containing most of the HCS or HGH coding nucleotide sequence were synthesized. Furthermore, new strains of microorganisms have been generated which contain the said genes and parts of genes. The nucleotide sequences were re-isolated after numerous replications in the host organism and their nucleotide sequence was found to be substantially identical to the sequence present in the parent organism.

Based on the genetic code, there is a finite array of nucleotide sequences encoding a given amino acid sequence. All of these equivalent nucleotide sequences are useful variants of the disclosed sequences because they all result in the same protein hormone having the same amino acid sequence in the course of transcription and translation in vivo. Consequently, all such variations are included within the scope of the invention.

The microorganism E. coli HCS X-1776 described here has been deposited with the ATCC under the accession number 31391. This microorganism carries a DNA sequence encoding the majority of human HCS. The DNA sequence is carried by a plasmid which has also been deposited with the ATCC and has the accession number 40002.