DD255359A5 - METHOD FOR DETERMINING A SPECIFIC SINGLE-TERM POLYNUCLEBIDE OF UNKNOWN SEQUENCE - Google Patents

Abstract

A method for the determination of a specific single-stranded polynucleotide of unknown sequence in a heterogeneous cellular or viral sample including multiple single-stranded polynucleotides, characterized in that (a) a mixture of labeled single-stranded polynucleotide probes with equally varying sequences of the bases is prepared, each of the probes potentially forming a sequence of Bases is complementary, which is the alleged only once existing copy to the polynucleotide to be determined; (b) fixing the sample on a solid substrate; (c) treating the substrate with the sample fixed thereon to attenuate further binding of polynucleotides thereto, except by hybridization to polynucleotides in the sample; (d) contacting the treated substrate with the sample fixed thereon in a volatile manner with said mixture of labeled probes under conditions which only facilitate hybridization between fully complementary polynucleotides; and (e) determining the specific polynucleotide by observing the occurrence of a hybridization reaction between it and a completely complementary probe from the mixture of labeled probes.

Description

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Field of application of the invention

The invention relates to a method for producing a polypeptide. In general, the invention relates to the manipulation of genetic materials, in particular to recombinant methods that enable the production of polypeptides that have partial or total primary structural organization and / or one or more of the biological properties of naturally occurring erythropoietin.

Characteristic of the known technical solutions

A. Manipulation of genetic materials

Genetic materials can be broadly defined as those chemical substances that program and direct the production of cellular components and viruses, and direct the response of cells and viruses. A long-chain polymeric substance known as deoxyribonucleic acid (DNA) contains the genetic material of all living cells and viruses, with the exception of certain viruses programmed by the Rebonucleic Acids (RNA). The repeating units in DNA polymers are four different nucleotides, each of which consists of either a purine (adenine or guanine) or a pyrimidine (thymine or cytosine) found on a deoxyribose sugar to which a phosphate group is attached. The attachment of nucleotides in linear form is via the fusion of 5'-phosphates of one nucleotide to the 3'-hydroxy group of another. Functional DNA occurs in the form of stable double-stranded assemblies of single-stranded nucleotides (known as deoxyoligonucleotides) resulting from hydrogen bonding between purine and pyrimidine bases (eg, "complementary" assemblages found between either adenine [A] and thymine [T By convention, the nucleotides are designated by the names of their constitutional purine or pyrimidine bases, and the complementary aggregations of the nucleotides in the double-stranded DNA (eg, -AT and GC). are called "base pairs". Ribonucleic acid is a polynucleotide consisting of adenine, guanine, cytosine and uracil (U) instead of thymine attached to ribose and a phosphate group.

In summary, the programming function of DNA is generally accomplished by a process in which specific DNA nucleotide sequences (genes) are "transcribed" into relatively unstable messenger RNA (mRNA) polymers, conversely, which serves as a template for the formation of structural amino acid regulatory and catalytic proteins. This mRNA "translation" process involves the operation of small RNA strands (tRNA) that transport and align single amino acids along the mRNA strand to allow the formation of polypeptides in proper amino acid sequence. The mRNA "message", derived from the DNA and forming the basis for tRNA support and the orientation of any given one of the twenty amino acids for polypeptide "expression", is in the form of triple "lodons" - successive moieties of the three nucleotide bases In this sense, the formation of a protein is the last form of "expression" of the programmed genetic message present in the nucleotide sequence of a gene. "Promoter" DNA sequences usually "precede" a gene in a DNA polymer and provide a site for initiation of transcription into mRNA. "Regulator" DNA sequences, also usually upstream of the (e.g., precursor) gene in a given DNA polymer, bind proteins that determine the rate (or rate) of transcription initiation.

Commonly referred to as a "promoter / regulator" or "control" DNA sequence, these sequences, which precede a selected gene (or set of genes) in a functional DNA polymer, work together to determine whether transcription (and eventually expression) of a gene takes place. DNA sequences "following" a gene in a DNA polymer and providing a signal for cessation of transcription into mRNA are referred to as transcriptional terminator sequences.

The focus of microbiological processes in the last decade has been the attempt to produce industrially and pharmaceutically significant substances using organisms which either initially did not contain genetically encoded information regarding the desired product in their DNA or (in the case of human cells in culture) usually no chromosomal gene express corresponding levels. Simplified, a gene that specifies the structure of a desired polypeptide product is either isolated from a "donor" organism or chemically synthesized and then stably introduced into another organism, preferably a self-replicating, multicellular organism such as bacteria , Yeast or mammalian cells in culture. Once this has been done, the existing mechanism for gene expression in the "transformed" or "transferred" microwell host cells is used to construct the desired product using the exogenous DNA as a template for the transcription of mRNA which is then expressed in a continuous sequence of amino acid residues is translated.

A number of patent and literary publications on recombinant DNA technology for the isolation, synthesis, purification, and amplification of genetic materials for use in the transformation of selected host organisms are in the art, for example, U.S. Patent 4,237,224 (Cohen et al. ) the transformation of unicellular host organisms with "hybrid" viral or circular plasmid DNA, including selected exogenous DNA sequences. Among the procedures of the Cohen patent is the preparation of a transformation vector by enzymatic cutting of viral or circular plasmid DNA to form linear DNA strands. Selected foreign ("exogenous" or "heterologous") DNA strands, which usually include sequences encoding the desired product, are prepared in linear form through the use of similar enzymes. The linear viral or plasmid DNA is incubated with the foreign DNA in the presence of ligating enzymes capable of effecting a recovery process, and "hybrid" vectors are formed, including the selected exogenous DNA segment, "Spliced" into the viral or circular DNA plasmid.

Transformation of compatible unicellular host organisms with the hybrid vector results in the production of replicated copies of the exogenous DNA in the host cell population. In many cases, the desired result is simply the amplification of the foreign DNA, and the recovered "product" is the DNA. It has recently been reported that the ultimate goal of the transformation is the expression by the host cells of the exogenous DNA in the form of a large-scale synthesis of isolatable amounts of commercially significant, coding therefor protein or polypeptide fragments by the foreign DNA, see also, for example, US Patent 4264731 (Shine), 4273875 (Manis), 4293652 (Cohen) and EP-OS 093619, published on 9 November 1983.

The development of specific DNA sequences for splicing into DNA vectors is accomplished via a variety of techniques, largely depending on the degree of "foreignness" of the "donor" to the intended host and the size of the polypeptide to be expressed in the host. On the danger of over-simplification, it can be stated that three alternative principal methods can be used: (1) the "isolation" of the double-stranded DNA sequence from the genomic DNA of the donor; (2) the chemical production of a DNA sequence containing a And (3) the in vitro synthesis of a double-stranded DNA sequence by enzymatic "reverse transcription" of mRNA isolated from donor cells. The latter methods, which include the formation of a DNA "complement" of mRNA, are commonly referred to as "cDNA" methods.

The production of DNA sequences has recently been termed the method of choice when the entire sequence of amino acid residues of the desired polypeptide product is known. DNA production method of the co-pending, co-pending U.S. Patent Application No. 483481 to Alton et al. (filed on April 15, 1983 and corresponding to PCT US83 / 00605, published November 24, 1983 as WO 83/04053 see, for example, further means of achieving such highly desirable results as: providing alternating codons recently found in genes Avoiding the presence of untranslated "intron" sequences (commonly found in human genomic DNA sequences and, for example, in human genomic DNA sequences, which are highly expressed in the host organisms selected for expression (e.g., provision of yeast or ecoli "preferred" codons) mRNA transcripts thereof) that are not readily processed by procaryotic host cells; avoiding the expression of unwanted "leader" polypeptide sequences, which are commonly encoded by genomic DNA and cDNA sequences, but not recently readily by the bacterial or polypeptide of interest Cleaved from yeast host cells, providing a slight insertion of the D NA into suitable expression vectors in association with desired promoter / regulator and terminator sequences; and providing a lightweight construction of genes encoding polypeptide fragments and analogs of the desired polypeptides.

• -3- 255 3B9

If the entire sequence of amino acid residues of the desired polypeptides is not known, direct production of the DNA sequences is not possible, and the isolation of DNA sequences encoding the polypeptide by a cDNA method becomes the method of choice, regardless the potential decreases in scope of the set of expression vectors capable of realizing a high level of microwave expression reported above. Standard methods for isolating cDNA sequences of interest include the preparation of plasmid-derived cDNA "libraries" derived from reverse transcription of mRNA rich in donor cells selected to be responsible for a high level of expression of genes (e.g. Libraries of cDNA derived from pituitary cells expressing relatively large amounts of growth hormone products.

Where substantial amounts of the amino acid sequences of the polypeptide are known, labeled single-stranded DNA probing sequences can be used which duplicate a sequence presumably present in the "target" cDNA in DNA / DNA hybridization procedures performed on cloned copies of the cDNA which have been degraded into a single-stranded form (see in general the disclosure and discussion of prior art in Weissman et al., U.S. Patent 4,344,443, and the recent demonstration of the use of long oligonucleotide hybridization probes described by Wallace et al. in Nuc. Acids Res. 6, pp. 3543-3557 [1979] and Reyes et al., in PNAS [USA], 79, pp. 3270-3274 [1982] and Jaye et al in Nuc. Acids Res. 11, p See, also, U.S. Patent No. 4,358,535 [Falkow et al.], Which relates to DNA / DNA hybridization methods for effecting diagnosis; Published European Patent Applications No. 2325-2335 [1983]. 0070685 and 0070687 concerning the light-emitting labels on single-stranded polynucleotide probes; Davis et al. "A Manual for Genetic Engineering, Advanced Bacterial Genetics", Cold Spring Harbor Laboratory, Cold Spring Harbor, NY [1980], pages 55-58 and 174-176, for colony and blood Plaque hybridization techniques; and New England Nuclear [Boston, Mass.] Brochures for "Gene Screen" hybridization transfer membrane materials with instructions for the transfer and hybridization of DNA and RNA, Catalog No. NEF-972).

Among the most significant recent advances in hybridization methods for the screening of recombinant clones is the use of labeled mixed synthetic oligonucleotide probes, each of which is potentially the full complement of a specific DNA sequence in the hybridization probe, including a heterogeneous mixture of single-stranded DNA's or RNA's. These procedures have been found to be particularly useful in detecting cDNA clones derived from such sources as to allow extremely low levels of mRNA sequences for the polypeptides of interest. In summary, the use of harsh hybridization conditions directed to the avoidance of non-specific binding may e.g. For example, for the autoradiographic visualization of a specific cDNA clone in the case of hybridization of the target DNA to this single probe within the mixture which is its complete gag. See generally, Wallace et al., Nucl. Acids Res. 9, pp. 879-897 (1981); Suggs et al., P.N.A.S. (USA), 78 pp. 6613-6617 (1981); Choo et al., Nature, 299, p. 178-180 (1982); Kurachi et al., P.N.A.S. (USA), 79, pp. 6461-6464 (1982); Ohkubo et al., P.N.A.S. (USA) 80, p.2196-2200 (1983); and Kornbluth et al., P.N.A.S. (USA) 80, pp. 3218-3222 (1983). Generally, the mixed probe methods of Wallace et al. (1981), p. o. by various investigators expanded to the point where reliable results were presumably obtained in cDNA clone isolation using a 32-part mixed pool of 16-base-long (16-mer) oligonucleotide probes of uniform, distinct DNA sequences with a single 11-mer to effect a two-site "positive" confirmation of the presence of cDNA of interest. See Singer-Sam et al., P.N.A.S. (USA), 80, p.802-806 (1983).

The use of genomic DNA isolates is the last common of the three above-mentioned methods for the development of specific DNA sequences for use in recombinant methods. This is particularly true in the field of recombinant methods directed to securing microwell expression of mammalian polypeptides and, in principle, does justice to the complexity of mammalian genomic DNA. Thus, while providing reliable procedures for the development of phage-born libraries of human and other mammalian genomic DNA (see, for example, Lawn et al., Cell 15, pp. 1157-1174 [1978] for methods for generating a human gene library Kam et al., PNAS [USA] 77, pp. 5172-5176 [1980] for a human gene library based on alternative restriction endonuclease fragmentation method; and Blattner et al., Science, 196, p. 161-169 [1977], which describes the construction of a bovine gene library), there are relatively few successful attempts to use hybridization techniques to isolate genomic DNA in the absence of extensive prior knowledge of amino acid or DNA sequences.

As an example, Fiddesetal., J. Mol. And Appl. Genetics, 1, pp. 3-18 [1981] reported the successful isolation of a gene encoding the human hypophyseglycoprotein hormone alpha subunit from the Maniatis library by using a full-length probe, a full 621 base pair Fragments of a previously isolated alpha subunit cDNA sequence.

As another example, et al., P.N.A.S. (USA), 80, pp. 1531-1535 (1983) for the isolation of human genomic clones for human HLA-DR using a 175 base pair synthetic oligonucleotide. Finally, Anderson et al., P.N.A.S. (USA), 80, pp. 6838-6842 (1983) for the isolation of a genomic clone for a bovine pancreatic trypsin inhibitor (BPTI) using a single probe of 86 base pairs in length and constructed according to the known amino acid sequence of BPTI. The authors observe a determination of low prospects for the isolation of mRNA suitable for the synthesis of a cDNA library, because of apparently low levels of mRNA in initially "targeted" parotid gland and lung tissue, and then direct the chances of success to probing a gene mRNA. Library using a mixture of labeled probes with the statement:

- "More generally, mixed sequence oligodeoxynucleotide probes were used to isolate protein genes of unknown sequence from cDNA libraries. Such probes are usually mixtures of 8-32 oligonucleotides, 14-17 nucleotides in length, representing any possible codon combination for a small stretch (5-6 residues) of the amino acid sequence. Under severe hybridization conditions that penalize non-correct base pair probes, these probes are capable of detecting the location of specific gene sequences in low to moderate complexity clone libraries. Nevertheless, because of their short length and heterogeneity, mixing probes often do not have the specificity required for probing sequences as a complex as a mammalian genome

is. This makes such a method unsuitable for isolation of mammalian protein genes if the corresponding mRNAs are not available. "

(End of quote).

Thus, there continues to be a need for improved methods that provide rapid and efficient isolation of cDNA clones in cases where little is known about the amino acid sequence of the polypeptide coding therefor and where "enriched" tissue material of mRNA is not readily available for use Such improved methods would be particularly useful if they were to be useful for the isolation of mammalian genomic clones where only dispersed information is available on the amino acid sequences of the polypeptides coding therefor by the genes of interest

 B. Erythropoietin as a polypeptide of interest

Erythropoiesis, the production of red blood cells, occurs continuously during human life to compensate for cell degeneration. The erythrocyte formation is a very well-controlled physiological mechanism that allows a sufficient number of red blood cells to be available in the blood to supply the tissue with oxygen, but not so much that the cells would hinder circulation. The formation of red blood cells takes place in the spinal cord and is controlled by the hormone erythropoietin.

Erythropoietin, an acidic glycoprotein of about 34,000 daltons in molecular weight, can exist in three forms: α, β, and asialo. The a and / or 3 forms differ slightly in the carbohydrate components but have the same efficacy, biological activity and molecular mass. The asialo form is an a- or / 3-form in which the terminal carbohydrate (sialic acid) is removed. Erythropoietin is present in very low concentrations in the plasma when the body is in a healthy state, with the tissue retaining sufficient oxygen over the existing number of erythrocytes. This normal low concentration is sufficient to stimulate the replacement of red blood cells, which usually precipitate through aging. ,

The amount of erythropoietin in the circulation increases under the conditions of tissue oxygen deficiency (hypoxia) when oxygen transport through the blood cells in the bloodstream is reduced. Hypoxia can be caused by loss of large quantities of blood due to hemorrhage, destruction of red blood cells by overdose of radiation, reduction of high levels of oxygen intake or prolonged unconsciousness, or by various forms of anemia. In response to tissue hypoxic stress, erythropoietin increases red blood cell production by stimulating the conversion of simple precursor cells in the spinal cord into pro-erythroblasts that later mature, synthesize hemoglobin, and are released into the bloodstream as red blood cells. If the number of red blood cells in the circulation is higher than required for the normal oxygen requirements of the tissue, the erythropoietin is reduced in the circulation. See in general. Testa et al., Exp. Hematol., 8 (Suppl.8), 144-152 (1980); Tong et al., J. Biol. Chem., 256 (24), 12666-12672 (1981); Gold Water, J. Cell. Physiol., 110 (Suppl.1), 133-135 (1982); Finch, Blood, 60 (6), 1241-1246 (1982); Sytowski et al., Expt. Hematol., 8 (Supp.8), 52-64 (1980); Naughton, Ann. CHn. Lab. Sci., 13 (5), 432-438 (1983); Weiss et al., Am. J. vet. Res., 44 (10), 1832-1835 (1983); Lappin et al., Esp. Hematol. 11 (7), 661-666 (1983); Bacin et al., Ann. N.Y. Acad. Sci., 414, 66-72 (1983); Murphy et al., Acta Haematologica Japonica, 46 (7), 1380-1396 (1983); Dessypris et al., Brit. J. Haematol., 56, 295-306 (1984); and Emmanoul et al. J. Physiol., 247 (1 Pt2), F168-76 (1984).

Since erythropoietin is essential in the process of red blood cell formation, the hormone has a potentially useful application in both the diagnosis and treatment of blood disorders characterized by low or defective red blood cell production. See generally Pennathur-Dasetal., Blood, 63 (5), 1168-71 (1984) and Haddy, at Jour. Ped. Hematol./Oncol. 4,191-196 (1982), which relates to erythroproietin for possible sickle cell anemia therapies, and Eschbach et al., J. Clin. Invest., 74 (2), pp. 434-441 (1984) describing a therapeutic regimen for uremic sheep based on in vivo responses to erythropietin-rich plasma infusions, and a proposed dose of 10 units EPO / kg per day for 15 to 40 days as a corrective of anemia associated with chronic renal failure. See also Cranes, Henry Ford Hosp., Med., 31 (3) 177-181 (1983)

 Recently, it has been estimated that the availability of erythropoietin in amounts is the anemia treatment of

1600000 people alone in the United States would allow. See e.g.. Morrison "Bioprocessing in Space

Overview "pages 557-571 in The World Biotech Report 1984, Vol.2: USA (Online Publications, New York, NY 1984) Recent studies have provided a basis for planning the efficacy of erythropoietin therapy in a variety of disease states, diseases and conditions haematological irregularity:

Vedovato, et al., Acta. Haematol. 71, 211-213 (1984) (beta-thalassemia); Vichinsky, et al., J. Pediatr., 105 (1), 15-21 (1984) (cystic fibrosis); Cotes, et al., Brit. J. Obstet. Gyneacol., 90 (4), 304-311 (1983) (pregnancy, menstrual cramps); Hage, et al., Acta. Pediatr. Scand., 72, 827-831 (1983) (early anemia of prematurity); Claus-Walker, et al. Arch. Phys. Med. Rehabil., 65, 373-374 (1984) (spinal cord injury) Dunn, et al., Eur. J. Appl. Physiol., 52, 178-182 (1984) (spaceflight); Miller, et al., Brit. J. Haematol., 52, 455-590 (acute blood loss); Udupa, et al., J. Lab. Clin. Med., 103 (4), 574-580 and 581-588 (1984); and Lipschitz, et al., Blood, 63 (3), 502-509 (1983) (aging); and Dainiak, et al., Cancer, 51 (6), 1101-1106 (1983) and Schwartz, et al., Otolaryngol., 109, 269-272 (1983) (various neoplastic disease states accompanied by abnormal erythropoiesia).

Previous attempts to recover erythropoietin in good yield from plasma or urine have been relatively unsuccessful. There are complicated and cumbersome laboratory procedures required, which generally resulted in the recovery of only very small amounts of impure and unstable erythropoietin containing extracts.

Initial attempts to isolate erythropoietin from urine have produced unstable, biologically inactive preparations of the hormone. US Pat. No. 3,865,801 describes a method for stabilizing the biological activity of a crude substance containing urine-derived erythropoietin. The resulting crude preparation containing erythropoietin apparently retains 90% of the erythropoietin activity and is stable.

Another method of purifying human erythropoietin from urine of patients with aplastic anemia is described by Miyake et al., J. Biol. Chem., Vol. 252, No. 15 (August 10, 1977), pp. 5558-5564. The seven-step procedure involves ion exchange chromatography, ethanol precipitation, gel filtration, and adsorption chromatography, yielding a pure erythropoietin preparation with a potency of 70,400 units / mg protein in 21% yield.

U.S. Patent No. 4,379,840 (Takezawa et al.) Describes methods for making "an erythropoietin product" from urine samples of healthy humans with weakly basic ion exchangers and states that the resulting low molecular weight product "has no inhibitory effects against erythropoietin."

GB 2,085,887 (Sugitomo et al.), Published May 6, 1982, describes a method for producing Hybfid human lymphoblastoid cells and reports prodrugs in the range of 3 to 420 units of erythropoietin per ml of cell suspension (distributed in the cultures After mammalian host propagation containing up to 10 ⁷ cells per ml, the best results reported to reach them were the production rate of erythropoietin at 40 to 4000 units / 10 ⁶ cells / 48 hours in vitro (See also U.S. Patent No. 4,377,513.) Numerous proposals have been made for the isolation of tissue erythropoietin, including neoplastic cells, but the yields were very low See, for example, Jelkman, et βΙ, Έχρΐ, Hematol., 11 (7), 581-588 (1983 Y Tambourin, et al., PNAS (USA), 80, 6269-6273 (1983); Katsucka, et al., Gann, 74, 534-541 (1983); Hagiwara et al., Blood, 63 (4), 828-835 (1984); and Choppin, et al., Blood 64 (2), 341-347 (1984). Other isolation methods used to obtain purified erythropoietin include immunological procedures. An anti-erythropoietin-directed polyclonal serum-derived antibody was developed by injecting an animal, preferably a rat or rabbit, with human erythropoietin. The injected human erythropoietin is recognized by the animal's immune system as a foreign antigenic substance and triggers the production of antibodies to the antigen. Different cells that respond to stimulation by the antigenic substance produce and release antibodies that are slightly different from those of other reacting cells. The antibody activity is retained in the animal's serum when the blood is extracted. While crude serum or antibody preparations purified as a serum immunoglobulin G fraction can then be used in assays to detect and complex with human erythropoietin, the materials have a significant drawback. This serum antibody, composed of all the different antibodies produced by single cells, is polyclonal in nature and will complex with components in crude extracts, unlike erythropoietin alone.

Also of interest to the background of the present invention are recent advances in the field of developing continuous cell cultures capable of producing a single type of antibody that is specifically immunologically reactive with a single antigenic determinant of a selected antigen. See, generally, Chisholm, High Technology, Vol.3, No. 1, 57-63 (1983).

Attempts have been made to use cell fusion and hybridization techniques to secrete "monoclonal" antibodies in the isolation and quantification of human erythropoietin, in short Lee-Huang, Abstract No.1463, Fed Proa, 41, 520 (1982). As a further example, a detailed description of the preparation and use of anti-erythropoietin moonclonal antibodies has been published by Weiss et al., PNAS (USA) 9, 5465-5469 (1982) See, Sasaki, Biomed., Biochim, Acta., 42 / 11 (12), 5202-5206 (1983), Yanagawa, et al., Blood 64 (2), 357-364 (1984), Yanagawa, et al., J. Biol. Chem., 259 (5), 2707-2710 (1984); and U.S. Patent 4,465,624.

Also of interest for the background of the invention are reports of the immunological activity of synthetic peptides which substantially double the amino acid sequences present in naturally occurring proteins, glycoproteins and nucleoproteins. In particular, relatively low molecular weight polypeptides have been shown to participate in immune responses that are similar in duration and extent to the immune responses of physiologically important proteins, such as viral antibodies, polypeptide hormones, and the like. Immune responses of such polypeptides also include provoking the formation of specific antibodies in immunologically active animals. See, e.g. Lemer et al., Cell, 23, 309-310 (1981); Ross, et al., Nature, 294, 654-656 (1981); Walter, et al., P.M. A.S. (USA), 77, 5197-5200 (1980); Lerner, et al .: P.N.A.S. (USA), 78, 3403-3407 (1981); Walter et al., P.N. (USA), 78, 48882-4886 (1981); Wong, et al., P.N. (USA), 78, 7412-7416 (1981); Green, et al., Cell, 28, 477-487 (1982); Nigg, et al., P.N.A.S. (USA), 79, 5322-5326 (1982); Baron, et al., Cell, 28, 395-404 (1982); Dreesman, et al., Nature, 295, 158-160 (1982); and Lerner, Scientific American, 248, no. 2,66-74 (1983). See also Kaiser, et al., Science, 223, pp. 249-255 (1984) for biological and immunological activities of synthetic peptides that approximately share the secondary structures of the peptide hormones but not their primary structural arrangement. The above studies, of course, relate to amino acid sequences of proteins other than erythropoietin, a substance for which no essential amino acid sequence has been previously described. In co-owned U.S. Patent Application No. 467244, filed on February 4, 1983 by J. Egrie, published August 22, 1984, EP-OS 0116446 discloses a mouse-mouse hybridoma cell line (ATCC HB 8209 ) which produces a highly specific, monoclonal, anti-erythropoietin antibody that is also specifically immunoreactive with a polypeptide consisting of the following sequence of amino acids: NHa-Ala-Pro-Pro-Arg-Leu-Ile-Cys Asp-Ser-Arg-Val-Leu-Glu-Arg-Tyr-Leu-Leu-Glu-Ala-Lys-COOH.

The polypeptide sequence is one attributable to the first twenty amino acid residues of mature human erythropoietin prepared by the method of Miyakeetal., J. Biol. Chem. 252, 5558-5564 (1977) and for which amino acid analysis was performed by gas-phase sequencer (Applied Biosystems, Inc.) according to the procedure of Hewick, M. et al., J. Biol. Chem., 256, 7990- 7997 (1981). See also Sue et al., Proc. Nat. Acad. Be. (USA), 80, p.3651-3655 (1983) regarding the development of polyclonal antibodies to a synthetic 26-mer based on a different amino acid sequence, and Sytowski et al., J. Immunol. Methods, 69, p. 181-186 (1984).

While polygonal and monoclonal antibodies, as described above, lead to very useful products for use in immunoassays for the determination and quantification of erythropoietin and are useful in the affinity purification of erythropoietin, it seems unlikely that these materials will readily isolate larger quantities of erythropoietin on a large scale can be used from mammalian material sufficient for further analysis, clinical investigation, and potentially far-reaching therapeutic use of the substance in the treatment of, for example, chronic kidney disease where diseased tissue is unable to sustain the production of erythropoietin. It is therefore believed in the art that the best prospects for fully labeling mammalian erythropoietin and providing it with large amounts for potential diagnostic and clinical use is to successfully use the recombinant techniques to allow for large scale microbial synthesis of the compound.

While it appears that substantial progress has been made in attempting to isolate DNA sequences encoding human and other mammalian species erythropoietin, it does not appear to have been so successful. This is principally due to the lack of tissue material, particularly human tissue material, which is mRNA-enriched and would allow to construct a cDNA library from which an erythropoietin-encoding DNA sequence could be isolated by standard techniques. In addition, the sequence of amino acid residues of erythropoietin is so little known that it is not possible, for example, to construct long polynucleotide probes capable of reliable use in DNA / DNA hybridization screening of cDNA and in particular genomic DNA libraries to become.

By way of illustration, the 20 amino acid sequence used to produce the above-mentioned monoclonal antibody produced by ATCC No. HB 8209, no construction of a unambiguous 60 base pair oligonucleotide probe as described by Anderson et al., Ibid. described way. It is estimated that humanity for erythropoietin appears as a "single copy gene" within the human genome and, in any event, the genetic material encoding human erythropoietin is likely to be less than 0.00005% of the total human genomic DNA that would be present in a gene library.

To date, the most successful of the literature-based attempts at recombinant-related methods of providing DNA sequences for use in microwell expression of isolatable amounts of mammalian erythropoietin have come to a head. As an example, Farber et al., Exp. Hematol. 11, Supp. 14, Abstract 101 (1983) on the extraction of mRNA from kidney tissue from phenylhydrazine-treated baboons and the injection of mRNA into Xenopus laevis oocytes with the rather volatile result of the in vitro production of a mixture of "translation production", including biological properties of Recently, Farber et al., Blood, 62, No. 5, Suppl. No. 1, Abstract 392 at page 122a (1983) reported the in vitro translation of human mRNA frog egg cell mRNA It was estimated that the resulting translation product mixture contained on the order of 220 (milliunits) (mU) of a translation product with the activity of erythropoietin per microgram of injected mRNA, while those levels were confirmed to be rather low by the in vitro translation of exogenous mRNA ( compared with the previously disclosed levels of baboon mRNA translation into the product sought) was ge believes that the results would confirm the human kidney as the site of erythropoietin expression and would permit the construction of an enriched human kidney cDNA library from which the desired gene could be isolated (see also Farber, Clin. Res., 31 [4], 769A [1983]). Since filing US Patent Application Nos. 561024 and 582185, a single report on cloning and expression has appeared, claiming to have obtained human erythropoietin cDNA in E. coli. A number of cDNA clones were inserted into E. coli plasmids and jS-lactamase fusion products were identified that were immunoreactive with a monoclonal antibody to an unspecified "epitope" of human erythropoietin. See Lee-Huang, Proc. Nat. Acad. Be. (USA), 81, Ss. 2708-2712 (1984).

Target of capture

The invention provides an improved method for the determination of a specific single-stranded polynucleotide of unknown sequence.

Explanation of the essence of the invention

The invention has for its object to provide a method for the determination of a specific single-stranded polynucleotides unknown sequence available.

The invention relates to a method for the determination of a specific single-stranded polynucleotides of unknown sequence in a heterogeneous cellular or viral sample including multiple single-stranded polynucleotides, characterized in that

(a) preparing a mixture of labeled single-stranded polynucleotide probes having uniformly varying sequences of the bases, each of said probes being potentially complementary to a sequence of bases which is said to be single-copy to the polynucleotide to be determined;

(b) fixing the sample on a solid substrate;

(c) treating the substrate with the sample fixed thereon to attenuate further binding of polynucleotides thereto, except by hybridization to polynucleotides in the sample;

(d) the treated substrate, with the sample fixed thereto, is made to be in volatile contact with said mixture of labeled probes under conditions which only facilitate hybridization between fully complementary polynucleotides; and

(e) determining the specific polynucleotide by observing the occurrence of a hybridization reaction between it and a fully complementary probe from the mixture of labeled probes.

The present invention illustrates monkey or human cloned DNA sequences and polypeptide sequences appropriately derived therefrom which are the primary structural arrangement of erythropoietins of their corresponding monkey or human origin.

Presenting the sequence of the amino acid residues of erythropoietin, the present invention contemplates the total and / or partial production of DNA sequences encoding erythropoietin and having such beneficial characteristics as including non-mammalian hosts for "preferred" codons for expression provided that sites are provided for cleavage by restriction endonuclease enzymes, and the further provision of additional initial, terminal or intermediate DNA sequences that facilitate the construction of easily expressible vectors Accordingly, the present invention relates to the preparation (and development by site-specific mutagenesis of cDNA and genomic DNA) of DNA sequences encoding microbial expression of polypeptide analogues or derivatives of erythropoietin

which differ from naturally occurring forms by the identity or location of the arrangement of one or more arnino acid residues (i.e., deletion analogs containing less than all of the EPO-specifying residues and / or substitution analogues wherein one or more of the specified residues is replaced by other residues and / or addition analogs wherein one or more amino acid residues are attached to a terminal or medial portion of the polypeptide); and participate in some or all of the properties of naturally occurring forms.

The new. DNA sequences of the invention contain all sequences necessary for safe expression in procaryotic Vi or eucaryotic host cells of polypeptide products which "comprise at least part of the primary structural arrangement and one or more of the biological properties of erythropoietin comprising: (a); the DNA sequences listed in Tables V and VI or their complementary strands;

(b) DNA sequences that hybridize (under hybridization conditions, as discussed herein or more stringent conditions) to DNA sequences as defined under (a), or to fragments thereof; and

(c) DNA sequences which, however, would hybridize with the degeneracy of the genetic code to (a) and (b) defined DNA sequences. Specifically included in part (b) are genomic DNA sequences that encode allelic variant forms of adenovirus human erythropoietin and / or decode other mammalian species of erythropoietin. Specifically included in part (c) are prepared DNA sequences that encode EPO, EPO fragments and EPO analogs whose DNA sequences may contain condons that facilitate the translation of messenger RNA into non-vertebrate hosts.

 Further assignable to the present invention is the class of polypeptides encoding part of the DNA complement to the upper strand of the human genomic DNA sequence of Table VI, i. "Complementary reverse proteins" as described previously by Tramontano et al. Nucleic Acids Research, 12, pp. 5049-5059 (1984).

The polypeptide products of the present invention can be "labeled" by covalent attachment to a detectable marker (e.g., radiolabeled with 125 I) to yield reagents used in the detection and quantification of erythropoietin in solid tissue and fluid samples such as blood or urine The DNA products of the present invention may also be labeled with detectable markers (such as radiotranslucent and non-isotopic markers such as biotin) and used in DN A hybridization procedures to determine the erythropoietin gene position and / or gene position of any related gene They can also be used to detect erythropoietin gene deficiencies based on DNA content and as a gene marker to identify adjacent genes and their inconsistencies.

As will be described in detail hereinafter, the present invention involves significant improvements in the detection methods of a particular single-stranded polynucleotide of unknown sequence in a heterogeneous cellular or viral sample containing multiple single-stranded polynucleotides, wherein

(a) preparing a mixture of labeled, single-stranded nucleotide probes having uniformly varying base sequences, each of said probes being potentially specifically complementary to a sequence of bases to be determined in the one or more sequences. Polynucleotide is believed to exist only once, (b) the sample is fixed on a solid substrate,

(c) treating the substrate with the sample thereon to prevent further binding of polynucleotides thereto, except by hybridization to polynucleotides in the sample; (d) - the treated substrate with the sample fixed thereon is volatile with said mixture labeled probes are contacted under conditions which facilitate hybridization only between fully complementary polynucleotides, and

(e) the specific polynucleotide is determined by observing the course of a hybridization reaction between it and a fully complementary probe within the mixture of labeled probes, as evidenced by the presence of a higher density of labeled material on the substrate at the site of the specific polynucleotide, as compared to one Background density of the labeled material resulting from nonspecific binding of labeled probes to the substrate.

The methods are particularly effective in situations requiring the use of 64,128,256,512,1024 or more mixed polynucleotide probes of 17 to 20 bases in length in DNA / DNA or RNA / RNA or DNA / RNA hybridizations.

As discussed below, the above improved methods have permitted the recognition of cDNA clones encoding erythropoietin originating from monkey species within a library made from monkey kidney anemic cell mRNA. In particular, a mixture of 128 evenly varied 20-mer probes based on ammocytic sequence information derived from sequencing fractions of human erythropoietin was used in colony hybridization procedures to add seven "positive" erythropoietin cDNA clones within a total of 200,000 colonies More notably, the practical use of the improved methods of the present invention allows the rapid isolation of three positive clones by screening T 500,000 phage plaques representing a human gene library This was achieved using the above mixture of 128 20-mer probes along with a second set of 128 17-mer probes based on an amino acid analysis of a different contiguous sequence of human erythropoietin The above-mentioned illustrative methods constitute the first known case of using multiple mixed oil oligonucleotide probes in DNA / DNA hybridization methods directed to the isolation of mammalian genome clones, and the first known case of using a mixture of more than 32 oligonucleotide probes in the isolation of cDNA clones. Numerous aspects and advantages of the invention will become apparent to those skilled in the art from the following detailed description, in which practical aspects of the invention will be elucidated in its presently preferred embodiments.

In accordance with the present invention, DNA sequences encoding some or all of the polypeptide sequence of human and monkey species erythropoietin (hereinafter sometimes referred to as "EPO") have been isolated and further characterized, the monkey or human DNA has become eucaryotic and procaryotic Expression, resulting in isolatable amounts of polypeptides that show biological (eg immunological) properties of naturally occurring EPO and both in vivo and in vitro biological activities of EPO.

The monkey species DNA was isolated from a cDNA library constructed with mRNA obtained from monkey kidney tissue in a chemically induced anemic state and its serum. after immunological determination contained high levels of EPO compared to normal monkey serum. Isolation of the desired cDNA clones containing DNA-encoding EPO was achieved using DNA / DNA colony hybridization using a pool of 128 mixed radiolabelled 20-mer oligonucleotide probes involving rapid screening of 200,000 colonies. The pattern of oligonucleotide probes was stationed on amino acid sequence information obtained by enzymatic fragmentation and sequencing of a small human EPO sample.

Human DNA was isolated from a human genomic DNA library. Isolation of clones containing EPO-encoding DNA was achieved by DNA / DNA plaque hybridization using the pool of 128 mixed 20-mer oligonucleotide probes listed above and a second pool of 128 radiolabelled 17-mer probes whose sequences are Amino acid sequence information obtained from a different human enzymatic EPO fragment.

Positive colonies and plaques were checked for accuracy by dideoxy sequencing of clonal DNA using a subset of 16 sequences within the pool of 20-mer probes, and selected clones were subjected to nucleotide sequence analysis which determined the derivation of primary structural identity of the EPOs encoded therewith Allowed polypeptides. The deduced polypeptide sequences showed a high degree of homology to each other and to a partial sequence generated by amino acid analysis of human EPO fragments.

A selected positive monkey cDNA clone and a selected positive human gene clone were each inserted into a "shuttle" DNA vector amplified in E. coli and used to culture mammalian cells. Cultured growth of transferred host cells resulted in preparations of the culture medium supernatant containing more than 300 oM EPO per ml of culture fluid.

embodiment

The following examples are illustrative of the invention and are specifically directed to methods previously performed for the recognition of EPO-encoding monkey cDNA clones and human genomic clones, to methods resulting in such recognition, and to sequencing.

Specifically, Example 1 is directed to the amino acid sequencing of human EPO fragments and the construction of mixtures of radiolabeled probes based on the results of this sequencing. Example 2 is generally directed to methods involving the detection of positive monkey cDNA clones and provides information for treating the animal and preliminary radioimmunoassay (RIA) analysis of animal sera. Example 3 is directed to preparation of the cDNA library, colony hybridization screening and verification of positive clones for correctness, DNA sequencing of a positive cDNA clone, and generation of a primary stem m match (amino acid sequence) information of the monkey. EPO polypeptide. Example 4 is directed to methods relating to the recognition of positive human genomic clones leading to information on the origin of the gene library, about plaque hybridization methods, and checking for the correctness of positive clones. Example 5 is directed to the DNA sequencing of a positive genomic clone and the generation of amino acid sequence information of the human EPO polypeptide, including a comparison of these with the monkey EPO sequence information.

example 1

A. Human EPO Fragment Amino Acid Sequencing

Human EPO was isolated from urine and subjected to tryptic degradation resulting in the development and isolation of 17 separate fragments in amounts of approximately 100 to 150 picomoles.

The fragments were arbitrarily assigned numbers and analyzed for amino acid sequences by microsequence analysis using a gas phase sequencer (Applied Biosystems) to obtain the sequence information shown in Table I. Single-letter codes were used therein, and "X" denotes a remainder that was not further specified.

Table I

Fragment No. Result sequence analysis

T4a A-P-P-R

T4b G-K-L-K

T 9 · A-L-G-A-Q-K

T13 V-L-E-R

T16 A-V-S-G-L-R

T18 L-F-R

T21 K-L-F-R

T25 Y-L-L-E-A-K

T26 a L-I-C-D-S-R

T26b L-Y-T-G-E-A-C-R

T 27 T-I-T-A-D-T-F-R

T28 E-A-I-S-P-P-D-A-A-M-A-A-P-L-R

T30 E-A-E-X-I-T-T-G-X-A-E-H-X-S-L-N-E-X-I-T-V-P

T31 V-Y-S-N-F-L-R

T33 'S-L-T-T-L-L-R

T 35 V-N-F-Y-A-W-K

T38 G-Q-A-L-L-V-X-S-S-Q-P-W-E-P-L-Q-L-H-V-D-K

B. Pattern and Construction of Oligonucleotide Probe Mixtures

The amino acid sequences listed in Table I were re-examined in connection with the degeneracy of the genetic code as to whether the mixed probe methods can be applied to DNA / DNA hybridization methods in cDNA and / or genomic DNA libraries. This analysis showed that within fragment # T35 there existed a series of 7 amino acid residues (Val-Asn-Phe-Tyr-Ala-Trp-Lys) which can only be characterized by being one of 128 possible DNAs Sequences that spans 20 base pairs are encrypted. Thus, a first set of 128 20-mer oligonucleotides was prepared by standard phosphoamidite methods (see, for example, Beaucage et al., Tetrahedron Letters, 22, pp. 1859-1862 (1981) on a solid support according to the sequence listed in Table II synthesized.

Table II rest - VaI Asn Phe Tyr Ala Trp Lys 3 ' CAA TTG AAG ATG CGA ACC TT-5 T A A A T G G C C

Further analysis showed that within the fragment T38, there existed a series of 6 amino acid residues (Gln-Pro-Trp-Glu-Pro-Leu) based on which to prepare a pool of 128 mixed oligonucleotide 17-mer probes could be listed in Table III below.

Table III rest Gin Per Trp Glu Per Leu 3 ' GTT GGA ACC CTT GGA GA-5 C T C T kA G G C C

The oligonucleotide probes were labeled at the 5 'end with gamma- ³² -p-ATP, 7 500-8000 cc / mmole (ICN) using T ₄ polynucleotide kinase (NEN). ,

Example 2 A. Monkey treatment procedure and RIA analysis

Female cynomolgus monkeys Macaca fascicularias (2.5-3 kg), one and a half to two years old) were treated subcutaneously with a pH7 solution of phenylhydrazine hydrochloride at a dose of 12.5 mg / kg on the first, third and fifth day. Before each injection, the hematocrit value was observed. On the seventh day, or at the time when the hematocrit level fell below 25% of the initial value, serum and kidneys were withdrawn following administration of 25 mg / kg doses of ketamine hydrochloride. The extracted materials were immediately frozen in liquid nitrogen and stored at -70 ⁰ C.

B. RIA for EPO

Radioimmunoassays were used to quantitatively determine EPO in samples according to the following procedures:

An erythropoietin standard or an unknown sample was incubated with antiserum for 2 hours at 37 ° C. After the two hour incubation, the test tubes were cooled in ice, ¹²⁵ L-labeled erythropoietin added and the tubes incubated at 0 ° C for at least 15 more hours. Each assay tube contained 500μΙ incubation mixture consisting of 50 μΙ diluted immune sera, lOOOOcpm ¹²⁵ l erythropoietin, δμΙΤ ^ 0 ^ βγΙοΙ and β250μΙ either of the EPO standards or the unknown sample, containing 0.1% BSA PBS was achieved the remaining volume , The antiserum used was the second test blood of a rabbit immunized with 1% pure human urinary erythropoietin preparation. The final dilution of antiserum in the assay was adjusted so that the antibody-bound ¹²⁵ I-EPO exceeded 10-20% of the total input counts. In general, this corresponded to a final dilution of the antiserum of 1: 50,000 to 1: 100,000.

The antibody-bound ¹²⁵ L erythropoietin was supplemented with 150 μ ^ βρί! A likes. After a 40 minute incubation, the samples were centrifuged and the pellets washed twice with 0.75 ml 10 mM Tris-HCl pH 8.2 containing 0.15 M NaCl, 2 mM EDTA and 0.05% Triton x-100. The washed pellets were counted in a gamma counter to determine the percentage of ¹²⁵ L erythropoietin binding. The counts bound by pre-immune sera were subtracted from all totals to correct nonspecific precipitation. The erythropoietin content of the unknown samples was determined by comparison with the standard curve.

The above procedure was applied to monkey serum obtained in part A (s.c.) and also to the untreated monkey serum. Normal serum levels were tested and showed approximately 36mU / ml, while treated monkey serum contained from 1000 to 1700mU / ml.

Example 3 A. Construction of the Apical cDNA Library

Messenger RNA was isolated from normal and anemic monkey kidneys using the guanidine thiocyanate method of Chirgwin et al., Biochemistry, 18, p. 5294 (1979), and the poly (A) ⁺ mRNA was separated by two passes of oligo (dT). Cellulose column chromatography purified in "Maniatis" et al., "Molecular Cloning, A Laboratory Manual", pp. 197-198 Cold Springs Harbor Laboratory, Cold Springs, Harbor, NY, 1982). The cDNA library was prepared according to a modification of the general procedures of Okayama et al., Mol. And Cell. Biol., 2, pp. 161-170 (1982). The key features of the presently preferred methods are the following:

(1) pUC8 was used as the sole vector, cut with Pst I and then provided with a tail of OligodT of 60 to 80 bases in length;

(2) Hinc II digestion was used to remove the oligo dT tail from one end of the vector;

(3) the first strand synthesis and tailing of oligo dG was done according to the published procedure

(4) Bann HI digestion was used to remove the oligo dG tail from one end of the vector; and

(5) Replacement of the RNA strand by DNA in the presence of two linkers (GATCTAAAGACCGTCCCCCCCC and ACGGTCTTTA) in a three-fold molar excess over the oligo dG tailed vector.

B. Colony hybridization method for screening the monkey cDNA library

Transformed E. coli was seeded at a density of 9000 colonies per 10 x 10 cm plate on nutrient medium plates containing 50 micrograms / ml ampicillin. Gene Screen filters (New England Nuclear Catalog No. NEF-972) were pre-wetted on a BHI-CAM plate (Bacto Brain Heart Infusion 37g / L, casamino acids 2g / L and Agar 15g / L, 500 micrograms / ml chloramphenicol) and used to lift the colonies off the plate. The colonies were grown in the same medium for 12 hours to amplify the plasmid copy number. The amplified colonies (colony side above) were treated by placing the filters in rows on 2 pieces Whatman 3 MM paper, saturated with the following solutions in each case:

(1) 50 mM glucose-25 mM Tris-HCl (pH 8.0) - 10 mM EDTA (pH 8.0) for 5 minutes;

(2) 0.5M NaOH for ten minutes; and

(3) 1.0M Tris HCl (pH 7.5) for three minutes

The filters were then air dried in vacuo at 8 TOT for two hours.

By treatment with a solution containing 50 micrograms / ml of protease enzyme in buffer K [0.1 M Tris-HCl (pH 8.0) -0.15 NaCl

10 μM EDTA (pH 8.2) -0.2% SDS], the filters were then subjected to protease K degradation. Each filter have been specifically added to 5ml of the solution, and allowed the reduction at 55 ⁰ C for 30 minutes vorsieh go. Thereafter, the solution was removed.

The filters were then incubated with 4 ml of a pre-hybridization buffer (5 χ SSPE - 0.5% SDS -100 micrograms / ml SS E. coli DNÄ

- 5 χ BFP). The pre-hybridization treatment was carried out at 55 ^° C., generally for 4 hours or longer. Thereafter, the pre-hybridization buffer was removed.

The hybridization procedure was performed in the following manner. To each filter was added 3 ml of hybridization buffer (5 χ SSPE - 0.5% SDS -100 micrograms / ml yeast tRNA) containing 0.025 picomoles of each of the 128 probe sequences of Table II (the total mixture was referred to as EPV mixture) and the filters were kept at 48 ° C for 20 hours. This temperature was 2 ° C lower than the lowest of the calculated dissociation temperatures (Td) calculated for any of the probes.

After hybridization, the filters were washed three times for ten minutes on a shaker with 6 x SSC-0.1% SDS at room temperature and washed two to three times with 6 x SSC-1% SDS at the hybridization temperature (48 ° C). Autoradiography of the filters revealed seven positive clones among 200,000 colonies screened. The initial sequence analysis of one of the putative monkey cDNA clones (designated clone 83) was made for verification purposes by a modification of the procedure of Wallace et al., Gene, 16, pp. 21-26 (1981). The plasmid DNA of the monkey cDNA clone 83 was linearized by degradation with Eco Rl and denatured by heating in a boiling water bath. The nucleotide sequence was determined by the dideoxy method of Sanger et al., P.I.M. A. S (USA), 74, p. 5463-5467 (1977). A subset of the EPV mixture of probes consisting of 16 sequences was used as a primer for the sequencing reactions.

C. Sequencing of monkey EPO cDNA

The nucleotide sequence analysis of clone 83 was performed by the method of Messing, Methods in Enzymology, 101, p.20-78 (1983). From Table IV, a preliminary restriction analysis of the approximately 1 600 base pair Eco RI / Hind III-cloned fragment of clone 83 can be seen. Approximate localizations of restriction endonuclease enzyme recognition sites were made by the number of bases 3 'to the Eco RI site at the 5' end of the fragment. Nucleotide sequencing was performed by sequences of the individual restriction fragments with the intention of adapting overlapping fragments. For example, analysis of nucleotides in a restriction fragment determined the overlap of sequence information C113 (Sau at -111 / Sma I at ~ 324), and reverse order sequencing of fragment C73 (Alu I at ~ 424 / BstEll at ~ 203.

Table IV

Restriction enzyme recognition site Approximate location (s)

EcoRI 1

Sau3A 111

Smal 180

BsTEII, '203

Smal 324

Kpnl 371

Continuation of Table IV

Restriction enzyme recognition site Approximate location (s)

Rsal 372

AIuI 424

Pstl 426

AIuI 430

Hpal 466

AIuI 546

Pstl. 601

Pvull 604

AIuI 605

AIuI 782

AIuI 788

Rsal 792

Pstl - 807

AIuI 841

AIuI 927

Ncol 946

Sau3A 1014

AIuI 1072

AIuI 1115

AIuI 1223

Pstl 1301

Rsal 1343

AIuI 1384

Hindlll 1449

AIuI 1450

Hindlll 1585

Sequencing of approximately 1342 base pairs (within the region spanned by Sau 3A site 3 'to the Eco RI site and the Hind III site) and analysis of the reading frames allowed the preparation of the DNA and amino acid sequence formats shown in Table V are included. In the table, the putative initial amino acid residue of the aminoterminal mature EPO (verified by correlation of the above-mentioned sequence analysis of twenty amino terminal residues) was designated by the number +1. The presence of a methionine-specifying ATG codo (termed -27) "upstream" of the initial amino terminal alanine residue as the first residue designated for the amino acid sequence of the mature protein indicates the likelihood that EPO is initially expressed in the cytoplasm of a precursor form was expressed with a 27 amino acid leader region, which is excised before the mature EPO enters the circulation. Potential glycosylation sites within the polypeptide are indicated by asterisks. The estimated molecular mass of the translated region was 21,117 daltons, and the molecular mass of the 165 residues of the polypeptide that produced mature monkey EPO was determined to be 18,236 daltons.

Table V Translation of monkey EPO cDNA

Sow 3 A

CCCGGTGTGGTCACCGCGCGCCTAGGTCGCTGAG

-27 -20

Met GIy VaI His GIu Cys Pro AIa Trp

GGACCCCGGCCAGGCGCGGAGATG GGG GTG CAC GAA TGT CCT GCC TGG

-10

Leu Trp Leu Leu Leu Ser Leu VaI Ser Leu Pro Leu GIy Leu Pro

CTG TGG CTT CTC CTG TCT CTC GTG TCG CTC CCT CTG GGC CTC CCA

-1 +1

VaI Pro GIy AIa Pro Pro Arg Leu He Cys Asp Ser Arg VaI Leu

GTC CCG GGC GCC CCA CCA CGC CTC ATC TGT GAC AGC CGA GTC CTG

20

GIu Arg Tyr Leu Leu Giu Ala Lys Giu Ala Giu Asn VaI Thr Met

GAG AGG TAC CTC TTG GAG GCC AAG GAG GCC GAG AAT GTC ACG ATG 30

Gly Cys Ser Glu Ser Cys Ser Leu Asn Glu Asn lie Thr VaI Pro

GGC TGT TCC GAA AGC TGC AGC TTG AAT GAG AAT ATC ACC GTC CCA

50

Asp Thr Lys VaI Asn Phe Tyr AIa Trp Lys Arg Met GIu VaI GIy

GAC ACC AAA GTT AAC TTC TAT GCC TGG AAG AGG ATG GAG GTC GGG

60 70: '

Gin Gin Ala VaI GIu VaI Trp Gin GIy Leu AIa Leu Leu Ser GIu

CAG CAG GCT GTA GAA GTC TGG CAG GGC CTG GCC CTG CTC TCA GAA

80

Ala VaI Leu Arg GIy Gin Ala VaI Leu AIa Asn Ser Ser GIn Pro

GCT GTC CTG CGG GGC CAG GCC GTG TTG GCC AAC TCT TCC CAG CCT

90 100

Phe GIu Pro Leu GIn Leu His Met Asp Lys Ala He Ser GIy Leu

TTC GAG CCC CTG CAG CTG CAC ATG GAT AAA GCC ATC AGT GGC CTT

110

Arg Ser Thr Thr Leu Leu Arg AIa Leu GIy Ala Gin GIu AIa

CGC AGC ATC ACC ACT CTG CTT CGG GCG CTG GGA GCC CAG GAA GCC

120 130

He Ser Leu Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr He

ATC TCC CTC CCA GAT GCG GCC TCG GCT GCT CCA CTC CGA ACC ATC

140

Thr Ala Asp Thr Phe Cys Lys Leu Phe Arg VaI Tyr Ser Asn Phe

ACT GCT GAC ACT TTC TGC AAA CTC TTC CGA GTC TAC TCC AAT TTC

150 160

Leu Arg GIy Lys Leu Lys Leu Tyr Thr GIy GIu AIa Cys Arg Arg

CTC CGG GGA AAG CTG AAG CTG TAC ACG GGG GAG GCC TGC AGG AGA

165

GIy Asp Arg OP GGG GAC AGA TGA CCAGGTGCGTCCAGCTGGGCACATCCACCACCTCCCTCACCAACACTGCCTGTGCCACACCCTCCC

GAGAGCAGCTTTAAACTCAGGAGCAGAGACAATGCAGGGAAAACACCTGAGCTCACTCGGCCACCTGCAAAAGATGCAGGA CACGCTTTGGAGGCAATTTACCTG I I GCACCTACCATCAGGGACAGGATGACTGGAGAACTTAGGTGGCAAGCTGTGACTT CTCAAGGCCTCACGGGCACTCCCTTGGTGGCAAGAGCCCCCTTGACACTGAGAGAATATTTTGCAATCTGCAGCAGGAAAAATTA CGGACAGGTTTTGGAGGTTGGAGGGTACTTGACAGGTGTGTGGGGAAGCAGGGCGGTAGGGGTGGAGCTGGGATGCGAGT GAGAACCGTGAAGACAGGATGGGGGCTGGCCTCTGGTTCTCGTGGGGTCCAAGCTT

Hind III

The polypeptide sequence of Table V can be readily checked analytically for the presence of hydrophilic regions and / or secondary matching characteristics indicating potentially highly immunogenic regions, e.g. By the methods of Hopp et al., P.N. A.S. (USA), 78, p.3824-3828 (1981) and Kyte et al., J. Mol. Biol. 157, pp. 105-132 (1982) and / or Chou et al., Biochem., 13, p 222-245 (1974) and Advances in Enzymology, 47, pp. 45-47 (1978). Computer-aided analysis according to the method of Hopp et al. is possible via a program called PEP Reference Section 6.7, available from Intelligenetics, Inc. 124 University Avenue, Paolo Alto, California.

Example 4

A. Human Gene Library

A Ch4A phage-born human fetal liver gene library, prepared according to the methods of Lawn et al., Cell, 18, pp. 533-543 (1979), was obtained and provided for use in a plaque hybridization assay.

B. Plaque hybridization methods for screening a human gene library

Phage particles were lysed and the DNAs fixed on filters (50,000 plaques per filter) according to the procedures of Woo, Methods in Enzymology, 68, pp. 389-395 (1979), except for the use of Gene-Screene Plus filters (New England Nuclear Catalog No.NEF-976) and NZYAM plates (NaCl, 5g, MgCl ₂ -OH ₂ 0.2g, NZ-Amine A, 10g, yeast extract, 5g, casamino acids, 2g, maltose, 2g, and agar, 15g / l).

The air-dried filters were heat treated at 80 ^° C. for one hour and then degraded with proteinase K as in Example 3, Part B. The pre-hybridization was washed with 1 M NaCl - carried out over 4 hours or more 1% SDS buffer at 55 ⁰ C, after which the buffer was removed. Hybridization and post-hybridization washes were performed as described in Example 3, part B. Both the mixture of 128 20-mer probes designated EPV and the mixture of 12817-mer probes from Table HI (referred to as EPQ mixture) were used. Hybridization was performed at 48 ^° C. using the EPV probe mixture. The EPQ special mixture Hybridization was performed at 46 ⁰ C performed --4 degrees below the lowest calculated Td for components of the mixture. The hybridization probe for rehybridization was removed by boiling with 1 χ SSC-0.1% SDS for two minutes. Autoradiography of the filters revealed three positive clones (reactive with both probe mixtures) of 1500000 screened phage plaques. Verification for the correctness of the positive clones as EPO occlusions were obtained by DNA sequencing and electron micrographic visualization of the heteroduplex formation with the monkey cDNA of Example 3. This procedure also revealed multiple introns in the genomic DNA sequence.

Example 5

Nucleotide sequence analysis of one of the positive clones (designated AhE 1) was performed. The results obtained are listed in Table VI.

Table VI

AAGCTTCTGGGCTTCCAGACCCAGCTACTTTGCGGAACTCAGCAACCCAGGCATCTCTGAGTCTCCGCCCAAGACCGGGATGCC

ggacagccgccctctcctctaggcccgtggggctggccctgcaccgccgagcttcccgggatgaggxx

-27 -24

Met GIy VaI His

CCCGGTGACCGGCGCGCCCCAAGTCGCTGAGGGACCCCGGCCAAGCGCGGAG ATG GGG GTG CAC G · GTGAGTACTCGCGGGCTGGGCGCTCCCGGCGGCCGGGTTCCTGTTTGAGCGGGGATTTAGCGCCCCGGCTATTGGCCAAGAGG tggctgggttcaaggaccggcgacttgtcaaggaccccggaagggggaggggggtggggcagcctccacgtgccgcgggga

CTGTGCAGTTGTGTCGTTGTCAGTGTCTCG I.S. TTGCACACGCACAGATCAATAAGCCAGAGGCAGCACCTGAGTGCTTGCATG

GTTGGGACAG GCCTGACTCT

GIy GGC -23 GIu AA Cys TGT Pro CCT -20 AIa GCC Leu CTG GIu GAG Leu CTC Pro CCA VaI GTC Leu CTG Leu CTG Arg AGG Tyr TAC Leu CTC Leu TTG

Me ATC

χ Asn

Cys TGT

CAGCCTGGCTATCTGTTCTAG -10

Leu Ser -Leu Pro CTG TCG CTC CCT

10

Asp Ser Arg VaI GAC AGC CGA GTC

26 Th r ACG

CACTTG GTTTG G G GTGGAGTTG G GAAG CTAG ACACTGroughTACATAAG AATAAGTCTGGTG GCCCCAAACCATACCTG AAA CTAGGCAAGGAGCAAAGCCAGCAGATCCTACGCCTGTGGGCCAGGG

Trp Leu Trp Leu Leu Leu Ser

GCC TGG CTG TGG CTT CTC CTG TCC

-1 +1

GIy AIa Pro Pro Arg Leu

GCC GCC CCA CCA CGC CTC

20

Giu Ala Lys Giu Ala Giu

Leu CTG

GAG GCC AAG GAG GCC GAG AAT ATC

27 30

Thr GIy Cys Ala GIu CCAGAGCCTTCAGGGACCCTTGACTCCCCGGGCTGTGTGCATTTCAG ACG GGC TGT GCT GAA

40 χ

His Cys Ser Leu Asn GIu - Asn He Thr VaI Pro Asp Thr Lys VaI Asn Phe Tyr CAC TGC AGC TTG AAT GAG AAT ATC ACT GTC CCA GAC ACC AAA GTT AAT TTC TAT 50 55

AIa Trp Lys Arg Met GIu GCC TGG AAG AGG ATG GAG GTGAGTTCCI I I I I I I I I I I I ICCTTTCTmTGGAGAATCTCATTTGCGAGCCTGAT

CCCCATCTCACAAACATTTAAAAAATTAGTCAG GTGAAGTG GTGCATG GTG GTAGTCCCAG ATATTTGG AAG GCTG AG GCGGG AGGATCGCTTGAGCCCAGGAATTTGAGGCTGCAGTGAGCTGTGATCACACCACTGCACTCCAGCCTCAGTGACAGAGTGAGGCC

AG G GTGACATG G GTCAG CTCG ACTCCCAG AGTCCACTCCCTGTAG

56 VaI

GIy Gin GIn

60 Ala

VaI GIu VaI Trp Gin GIy Leu AIa

70 Lei Leu

Ser GIu AIa

GTC GGG CAG CAG GCC GTA GAA GTC TGG CAG GGC CTG GCC CTG CTG TCG GAA GCT

80

90

Leu Arg GIy Gin AIa Leu Lei VaI Asn Ser Ser GIn Pro Trp GIu Pro Leu GTC CTG CGG GGC CAG GCC CTG TTG GTC AAC TCT TCC CAG CCG TGG GAG CCC CTG

100

GIn Leu His VaI Asp Lys Ala VaI Ser GIy Leu Arg Ser Leu Thr Thr Leu Leu CAG CTG CAT GTG GAT AAA GCC GTC AGT GGC CTT CGC AGC CTC ACC ACT CTG CTT 110 115

Arg AIa Leu GIy Ala GIn

CGG GCT CTG GGA GCC CAG GTGAGTAGGAGCGGACACTTCTGCTTGCCCTTTCTGTAAGAAGGGGAGAAGGGTC TTGCTAAGGAGTACAGGAACTGTCCGTATTCCTTCCCTTTCTGTGGCACTGCAGCGACCTCCT

116 120

Lys GIu Ala He Ser Pro Pro Asp Ala Ala Ser Ala Ala GTTTTCTCCTTGGCAG AAG GAA GCC ATC TCC CCT CCA GAT GCG GCC TCA GCT GCT

130 140

Pro Leu Arg Thr Thr Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg VaI Tyr Ser

CCA CTC CGA ACA ATC ACT GCT GAC ACT TTC CGC AAA CTC TTC CGA GTC TAC TCC

150 160

Asn Phe Leu Arg GIy Lys Leu Lys Leu Tyr Thr GIy GIu AIa Cys Arg Thr GIy

AAT TTC CTC CGG GGA AAG CTG AAG CTG TAC ACA GGG GAG GCC TGC AGG ACA GGG

Asp - Arg OP

GAC "AGA TGA CCAGGTGTGTCCACCTGGGCATATCCACCACCTCCCTCACCAACATTGCTTGTGCCACACCCTCCCCCGCCA

CGCTTTGGAGGCGATTTACCTGTTTTCGCACCTACCATCAGGGACAGGATGACCTGGAGAACTTAGGTGGCAAGCTGTGACTTCT GGGCTGGCCTCTGGCTCTCATGGGGTCCAAGTTTGTGTATTCTCACCTATTGACAGACTGAAACACAATATGAC

In Table VI, the initial contiguous DNA sequence designates a 620 base top strand in which an apparently untranslated sequence immediately precedes a translated portion of the human EPO gene. In particular, it appears that the sequence comprises the 5 'end of the gene which directs to a translated DNA region coding for the first four amino acids (-27 to -24) of a leader sequence ("pre-sequence") Base pairs in the sequence that encoded the beginning of the leader sequence were not accurately determined and have therefore been termed "X". This is followed by an intron of approximately 639 base pairs (439 base pairs of which were sequenced and the remaining 200 base pairs of which were termed "IS") and immediately before a codon for glutamine designated as residue -23 of the translated polypeptide Exon sequence is considered to be coding for amino acid residues by an alanine residue (referred to as the + 1 residue of the amino acid sequence of mature human EPO) to the codon that specifies threonine at position +26, followed by a second intron consisting of 256 bases. as specifically indicated, this intron is followed by an exon sequence for amino acid residues 27 to 55, and then a third intron containing 612 base pairs begins.

The subsequent exon encodes residues 56 through 115 for the human EPO and then begins a fourth intron of 134 bases as listed. The fourth intron is followed by an exon coding for residues Nos. 116 to 166 and a stop codon (TGA). Finally, a sequence of 568 base pairs is identified in which an untranslated 3 'region of the human EPO gene appears, two base pairs, of which ("X") has not yet been accurately sequenced.

Table VI thus serves to establish the structural match (amino acid sequence) of mature human EPO containing 166 specific amino acid residues (estimated molar mass = 18,399). In addition, the table shows the DNA sequence encoding a 27 residue leader sequence along with 5 'and 3' DNA sequences which may be significant to promoter / operator functions of the human operon.

Sites for the potential glycosylation of the mature human EPO polypeptide are designated by asterisks in the table. It should be noted that the specific amino acid sequence of Table VI probably forms that of a naturally occurring allelic form of human erythropoietin. This position is supported by the results of ongoing urinary isolation of human erythoropoietin sequencing efforts which resulted in finding a significant number of erythropoietin molecules having a methionine at residue 126 versus a serine, as shown in the table.

The following Table VII illustrates the degree of polypeptide sequence homology between human and monkey EPO. Single-letter designations were used in the upper continuous row of the table to represent the deduced, translated polypeptide sequences of human EPO, starting with residue -27, and the bottom consecutive series showing the deduced polypeptide sequence from monkey EPO, starting with the number-1 polypeptide sequence. 27 denotes the rest. Asterisks indicate the sequence homologies. It should be noted that the deduced human and monkey EPO sequences have an "additional" lysine (K) residue at the (human) position 116. Comparison with Table VI shows that this residue is on the brink of a putative In addition, the presence of the lysine residue in the human polypeptide sequence was checked by sequencing a cDNA human sequence clone prepared from mRNA isolated from COS-1 cells spiked with the human genomic DNA of Example 7 (see below).

Table VII Comparison of human and monkey EPO polypeptides

-20 -10 +1 10 20 30 40

Human MGVHECPAWLWLLLSLSLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTK

XXXXXXXXXXXXXXXX XXXXXXXXXXXXXXXXXXXXXXXXXXXXXX XXXXXX X XXXXXXXXXXXXXX

Monkeys MGVHECPAWLWLLLSLVSLPLGLPVPGAPPRLICDSRVLERYLLEAKEAENVTMGCSESCSLNENITVPDTK

50 60 70 80 90 100 110

Human VNFYAWKRMEVGQQEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKE

XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX X XXXXX XXXXXX XXX XXXXX XXXXXXXXXX X

Table VIl (continued)

Monkeys VNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQAVLANSSQPFEPLQLHMDKAISGLRSITTLLRALGAQ-E

120 130 140 150 160

Human AISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR

XXX XXXXXXXXXXXXXXXXX XXXXXXXXXXXXXXXXXXXXXXX XXX

Monkey AISLPDAASAAPLRTITADTFCKLFRVYSNFLRGKLKLYTGEACRRGDR

A comparison of the activity of the recombinant monkey and human EPO materials against a standard of a naturally occurring human EPO and the results are shown graphically in FIG. Briefly, the results were expected to reveal that the recombinant monkey EPO competed significantly with anti-human EPO antibody, although it was unable to completely inhibit binding under the assay conditions. However, the highest inhibitory percentage for recombinant human EPO was close to that of the human EPO standard. The parallel location of the dose-response curves indicates immunological identity of the shared sequences (epitopes).

Example 11

The present example relates to the overall preparation by assembling the nucleotide bases of two structural genes encoding the human EPO sequence of Table VI and correspondingly enclosing "preferred" codons for expression in E. coli and yeast (s.cevevisiae) cells on.

Also described is the construction of genes that encode analogs of human EPO. Briefly, the procedure was as described in Alston et al., Supra (WO 83/04053). The genes were designed for an initial compilation of the component oligonucleotides in duplicate duplications, which were sequentially pooled into three separate sections. These sections were designed for ready amplification and, after removal from the amplification system, could be combined sequentially or by multiple fragment ligation into an appropriate expression vector.

The following Tables VIII through XIV illustrate the design and assembly of a prepared gene that encodes a human EPO translation product lacking any leader or presequence, but including an initial methionine residue at position -1. In addition, the gene in its essential part includes E. coli preferential codons, and the construction has therefore been termed the "ECEPO" gene.

Table VIII ECEPOSECTION 1 OLIGONUCLEOTIDES

1. AATTCTAGAAACCATGAG G GTAATAAAATA

2. CCATTATTTTATTACCCTCATGGTTTCTAG

3. ATGGCTCCGCCGCGTCTGATCTGCGAC

4. CTCGAGTCGCAGATCAGACGCGGCGGAG

5. TCGAGAGTTCTGGAACGTTACCTGCTG

6. CTTCCAGCAGGTAACGTTCCAGAACT

7. GAAGCTAAAGAAGCTGAAAACATC

8. GTGGTGATGTTTTCAGCTTCTTTAG

9. ACCACTGGTTGTGCTGAACACTGTTC

10. CAAAGAACAGTGTTCAGCACAACCA

11. TTTGAACGAAAACATTACGGTACCG

12. GATCCGGTACCGTAATGTTTTCGTT

Table IX ECEPO SECTION 1

Xfra l

EcoRI 1

AATTCTAG AAACCATGAG GGTAATAAAA Tj JaTGG CTCC GCCGCGTCTG GATC TTTGGTACTC CCATTATTTT ATTAG ^ GAGG CGGCGCAGAC

2 i

ATCTGCCACJT _- CGAGAGfTCT GGAACGTTAC CTGCTCJgAAG CTAAAGAAGC TAGACGCTGA GCTCJTCAAGA CCTTGCAATG GACGACUTT17 | GATTTCTTCG

TGAAAACATC. Iaccac tggtt gtgctgaaca ctgttcjtttg aacgaHaca

ACTTTTGTAG TGGTOACCAA CACGACTTGT GACAAGAAACI TTGCTTTTGT 8 ^r 10 '

Kpnl BamHI TTACGGTACC G AATGCCATGG CCTAG 12

Table X ECEPO SECTION 2 OLIGONUCLEOTIDES

1. AAT ^ CGGTACCAGACACCAAGGT

2. GTTAACCTTGGTGTCTGGTACCG

3. TAACTTCTACGCTTGGAAACGTAT

4. TTCCATACGTTTCCAAGCGTAGAA

5. GGAAGTTGGTCAACAAGCAGTTGAAGT

6. CCAAACTTCAACTGCTTGTTGACCAAC

7. TTGGCAGGGTCTGGCACTGCTGAGCG

8. GCCTCGCTCAGCAGTGCCAGACCCTG

9. AGGCTGTACTGCGTGGCCAGGCA

10. GCAGTGCCTGGCCACGCAGTACA

11. CTGCTGGTAAACTCCTCTCAGCCGT

12. TTCCCACGGCTGAGAGGAGTTTACCA

13. GGGAACCGCTGCAGCTGCATGTTGAC

14. GCTTTGTCAACATGCAGCTGCAGCGG

15. AAAGCAGTATCTGGCCTGAGATCTG

16. GATCCAGATCTCAGGCCAGATACT

Table XI ECEPO SECTION 2

EcoR I Kpn l ₁ _

A ATTCGGTACC AGACACCAAG Ga JTAAC TTCT ACGCTTGGAA ACGTAThGAA GCCATGG TCTGTGGTTC CAATfGiAAGA TGCGAACCTT TGCATAuUH!

g ± '

GTTGGTCAAC AAGCAGTTGA AGt JpTGG CAG GGTGTGGCAC TGCTGAGCCJA CAACCAGTTG TTCGTCAACT TCAAACCJGTC COAGACCGTG ACGACTCGCT 6 ^

^ J H J

GGCTGTACTG CGTGGCCAGG C 4CTGC TGGT AAACTCCTCT CAGCCG OjGGG

CCCJACATGAC GCACCGGTCC GTGACGIACCA TTTGAGGAGA GTCGGCACCC IO

13 15 BgI II BamH I

MCCGCTGCA GCTGCATGTT gac (aaagcag tatctggcct gagatctg

TOTGGCGACGT CGACGTACAA CTGTTTCGJTC ATAGACCGGA CTCTAGACCTAC

Table XII ECEPO SECTION 3

1. GATCCAGATCTCTGACTACTCTGC

2. ACGCAGCAGAGTAGTCAGAGATCTG

3. TGCGTGCTCTGGGTGCACAGAAAGAGG

4. GATAGCCTCTTTCTGTGCACCCAGAGC

5. CTATCTCTCCGCCGGATGCTGCATCT

6. CAGCAGATGCAGCATCCGGCGGAGA

7. GCTGCACCGCTGCGTACCATCACTG

8. ATCAGCAGTGATGGTACGCAGCGGTG

9. CTGATACCTTCCGCAAACTGTTTCG

10. ATACACGAAACAGTTTGCGGAAGGT

11. TGTATACTCTAACTTCCTGCGTGGTA

12. CAGTTTACCACGCAGGAAGTTAGAGT

13. AACTGAAACTGTATACTGGCGAAGC

14. GGCATGCTTCGCCAGTATACAGTTT

15. ATGCCGTACTGGTGACCGCTAATAG

16. TCGACTATTAGCGGTCACCAGTAC

Table XIII ECEPO SECTION 3

BamHI BglII GA TCCAGATCTCTG GTCTAGAGAC

ACTACfCTGC ITGCGT GCTCT GGGTGCACAG AAAGAGQCTA _- TCTCTCCGCC TGATGAGACG ACGC ^) CGAGA CCCACGTGTC TTTCTCCGAT AGjAGAGGCGG

L. 1.1

GGATGCTGCA TC TDCTGC AC CGCTGCGTAC CATCACT GPT GATA CCTTCC CCTACGACGT AGACGlÜfeTG GCGACGCATG GTAGTGACGA CMGGAAGG. · 6 8

11,

GCAAACTGTT TCG DGTAT ₁ A C TCTAÄCTTCC TGCGTGGTA IA ACTGA AACTG CGTTTGACAiI AGCACATAItG AGATTGAAGG ACGCACCATT TGAOJTTTGAC

12

, 15 sai l

TATACTGGCG AAGCJATGCCG TACTGGTGAC CGCTAATAG ATATGACCGC TTCGTACΰφ ATGACCACTG GCGATTATC AGCT

u. Ü

Table XIV ECEPO GENE

Xbal ~ ^1. ]

mETALA

CTAG AAACCATGAG GGTAATAAAA TAATGGCTCC GCCGCGTCTG

TTTGGTACTC CCATTATTTT ATTACCGAGG CGGCGCAGAC

ATCTGCGACT CGAGAGTTCT GGAACGTTAC CTGCTGGAAG CTAAAGAAGC

TAGACGCTGA GCTCTCAAGA CCTTGCAATG GACGACCTTC GATTTCTTCG

TGAAAACATC ACCACTGGTT GTGCTGAACA CTGTTCTTTG AACGAAAACA

ACTTTTGTAG TGGTGACCAA CACGACTTGT GACAAGAAAC TTGCTTTTGT

TTACGGTACC AGACACCAAG GTTAACTTCT ACGCTTGGAA ACGTATGGAA

AATGCCATGG TCTGTGGTTC CAATTGAAGA TGCGAACCTT TGCATACCTT

GTTGGTCAAC AAGCAGTTGA AGTTTGGCAG GGTCTGGCAC TGCTGAGCGA

CAACCAGTTG TTCGTCAACT TCAAACCGTC CCAGACCGTG ACGACTCGCT

GGCTGTACTG CGTGGCCAGG CACTGCTGGT AAACTCCTCT CAGCCGTGGG

CCGACATGAC GCACCGGTCC GTGACGACCA TTTGAGGAGA GTCGGCACCC

AACCGCTGCA GCTGCATGTT GACAAAGCAG TATCTGGCCT GAGATCTCTG

TTGGCGACGT CGACGTACAA CTGTTTCGTC ATAGACCGGA CTCTAGAGAC

ACTACTCTGC TGCGTGCTCT GGGTGCACAG AAAGAGGCTA TCTCTCCGCC

TGATGAGACG ACGCACGAGA CCCACGTGTC TTTCTCCGAT AGAGAGGCGG

GCATGCTGCA TCTGCTGCAC CGCTGCGTAC CATCACTGCT GATACCTTCC

CCTACGACGT AGACGACGTG GCGACGCATG GTAGTGACGA CTATGGAAGG

GCAAACTGH TCGTGTATAC TCTAACTTCC TGCGTGGTAA ACTGAAACTG CGTTTGACAA AGCACATATG AGATTGAAGG ACGCACCATT TGACTTTGAC

Sal I

TATACTGGCG AAGCATGCCG TACTGGTGAC CGCTAATAG ATATGACCGC TTCGTACGGC ATGACCACTG GCGATTATCA GCT

In detail, Table VIII shows oligonucleotides used to generate Section 1 of the ECEPO gene, which encodes the amino terminal residues of human polypeptide. Oligonucleotides were doubled (1 and 2, 3 and 4, etc.) and the doublings were then ligated to arrive at ECEPO Section 1 as in Table IX. It should be noted that the unified section includes terminal single-stranded EcoRI and BamHI ends, that "downstream" of the single-stranded EcoRI end is an Xbal restriction enzyme recognition site, and that "upstream" of single-stranded BamHI end is a Kpml recognition site , Section 1 could be easily amplified using the M13 phage vector used to check the sequence of the section: some difficulties were encountered in isolating the section as an XbaI / KpnI fragment from RF DNA generated in E. coli, probably because of the Methylation of KpnI recognition site bases within the host. Single-stranded phage DNA was therefore isolated and translated into the double-stranded form in vitro by primer extension, and the desired double-stranded fragment was then readily isolated.

ECEPO gene sections 2 and 3 (Tables XI and XIII) were similarly constructed from the oligonucleotides of Tables X and XII. Each section was amplified in the M13 vector, which was used for sequence verification for accuracy, and was isolated from phage DNA.

As can be seen from Table XI, ECEPO Section 2 was constructed with single-stranded EcoRI and BamHI ends and could be isolated as a KpnI / BglII fragment. Similarly, ECEPO Section 3 was made with single-stranded BamHI and Sall ends and could be isolated from phage RF DNA as a Bgl H-Sal I fragment. The three sections prepared in this manner could be readily pooled into a contiguous DNA sequence (Table XIV) which encodes the entire human EPO polypeptide with an amino terminal methionine codon (ATG) for E. coli translation initiation.

It is further noted that "upstream" from the initial ATG is a series of base pairs that duplicate the ribosomal binding site sequence of the highly expressed E. coli OMP f gene.

To carry the ECEPO, any suitable expression vector can be used. The particular vector chosen for expression of the ECEPO gene as the "temperature-sensitive" plasmid pCFM536 - a derivative of plasmid pCFM414 (ATCC 40076) is described in copending U.S. Patent Application No. 636727 filed August 6, 1964 described by Charles F. Morris.

In particular, pCFM 536 was degraded with XbaI and HindIII; the large fragment was isolated and used in a two-part ligation with the ECEPO gene. Sections 1 (XbaI / KpnI), 2 (Kpml / Bgl III), and 3 (BglI / SalI) were previously correctly combined in M13, and the EPO gene was isolated from it as a single Xba I / Hind HI. Fragment.

To this fragment belonged a portion of the polylinker from the M13 and mp9 phage that spanned the Sal I to Hind HI sites therein. Control of expression in the resulting expression plasmid p536 was by means of a lambda P _L promoter, itself controlled by the Cn ₈ 57 repressor gene (as provided by E. coli strain K12 Htrp).

The above-prepared ECEPO gene could be variously modified to encode erythropoietin analogs such as [Asn ² , Pro ² to all ^e ] EPO and [His ⁷ ] hEPO as described below.

A. [Asn ² , Pro ² to He ⁶ ] hEPO

Plasmid 53b carrying the ECEPO-produced gene of Table XIV as an XbaI to HindII insert was digested with HindIH and XhoI. The latter endonuclease cuts the ECEPO gene at a single 6 base pair recognition site spanning the last base of the Asp ⁸ encoding codon to the second base of the Arg '° codon.

An Xba I / Xho I "linker" sequence was prepared containing the following sequences:

XbaI mead + 1 2 7 δ 9 5'-CTAG ATG Ala Asn Cys Asp XhoI 3'-TAC GCT AAT TGC GAC-3 ' CGA TTA ACG 'CTG AGCT-5'

The XbaI / XhoI linker and the XhoI / HindIIIECEPO gene sequence fragment were inserted into the large fragment obtained from XbaI and HindIl digestion of plasmid pCFM 526 - a derivative of plasmid pCFM 414 (ARCC 40076). As described in co-pending U.S. Patent Application No. 6,36727, filed August 6, 1984, by Charles F. Morris, to generate a plasmid-born DNA sequence expressing E. coli expression of Met ^. Form of the desired analog coded.

B. [His ⁷ ] hEPO

Plasmid 536 was digested with Hind III and Xho 1 as in Part A above. An Xba I / Xho I linker was prepared which had the following sequence:

9Xhol

AGCT-5 '

The linker and the Xhol / Hind III ECEPO sequence fragment were then inserted into pCFM526 to generate a plasmid-supplied DNA sequence which encodes the E. coli expression of the Met ^ form of the desired analog.

XbaI mead + 1 2 3 4 CJL CJ) 7 δ ATG Ala Per Per bad Leu He His Asp 5'-CTAG GCT CCG CCA CGT CTG ATC CAT CAC-3 ' 3'-TAC CGA GGC GGT GCA GAC DAY GTA CTG

The construction of a prepared gene ("SCEPO") including the yeast preference codons is described in the following Tables XV to XXI As with the ECEPO gene, the entire construction involves the formation of three sets of oligonucleotides (Tables XV, XVII and XIX) which are formed into duplicates and were combined into sections (Tables XVI, XVIII, and XX) It is noted that the synthesis has been partially facilitated by the use of some suboptimal codons, both in SCEPO and ECEPO construction, ie, oligonucleotides 7-12 of Section 1 of both genes were identical, as were Section 2- oligonucleotides 1-6 in each gene.

Table XV SCEPO SECTION I OLIGONUCLEOTIDE

1. AATTCAAGCTTGGATAAAAGAGCT

2. GTGGAGCTCTTTTATCCAAGCTTG

3. CCACCAAGATTGATCTGTGACTC

4. TCTCGAGTCACAGATCAATCTTG

5. GAGAGTTTTGGAAAGATACTTGTTG

6. CTTCCAACAAGTATCTTTCCAAAC

7. GAAGCTAAAGAAGCTGAAAACATC

8. GTGGTGATGTTTTCAGCTTCTTTAG

9. ACCACTGGTTGTGCTGAACACTGTTC

10. CAAAGAACAGTGTTCAGCACAACCA

11. TTTGAACGAAAACATTACGGTACCG

12. GATCCGGTACCGTAATGTTTTCGTT

Table XVI SCEPO SECTION 1

EcoRI Hind III 1

CTtca aScTjtggata gt tggaacgtat

AAAGAGC JCC ACC AAGATTG ATCTGTGACT ( JgaGA GTTTT TTTCTCGAGG TGpTTCTAAC TAGACACTGA GCTCqCAAAA

5.1

GGAAAGATAC TTGTT dGAAG CTAAAGAAGC TGAAAACATC ACCAC TGGTT CCTTTCTATG AACAACCTTC] GATTTCTTCG ACTTTTGTAG TGGTqACCAA

9 H Kpnl BaEiHI

GTGCTGAlCA CTGTTCjTTTGiUCGAAAACA TTACGGTACC G CACGACTTGT GäCAAGAAAC TTGCTTTTGT AATGCCATGG CCTAG

12

Table XVII SCEPO SECTION 2 OLIGONUCLEOTIDE

1. AATTCGGTACCAGACACCAAGGT

2. GTTAACCTTGGTGTCTGGTACCG

3. TAACTTCTACGCTTGGAAACGTAT

4. TTCCATACGTTTCCAAGCGTAGAA

5. GGAAGTTGGTCAACAAGCAGTTGAAGT

6. CCAAACTTCAACTGCTTGTTGACCAAC

7. TTGGCAAGGTTTGGCCTTGTTATCTG

8. GCTTCAGATAACAAGGCCAAACCTTG

9. AAGCTGTTTTGAGAGGTCAAGCCT

10. AACAAGGCTTGACCTCTCAAAACA

11. TGTTGGTTAACTCTTCTCAACCATGGG

12. TGGTTCCCATGGTTGAGAAGAGTTAACC

13. AACCATTGCAATTGCACGTCGAT

14. CTTTATCGACGTGCAATTGCAA

15. AAAGCCGTCTCTGGTTTGAGATCTG

16. GATCCAGATCTCAAACCAGAGACGG

Table XVII SCEPO SECTION 2

Kpn I EcoRI 1

aTTTCGGTACC AGACACCAAG tlCCATGG TCTGTGGTTC

1 year

gt ttaact tcl ¹ acgcttggaa acgtaq jggaa gttggtcaac aagctgttga

caattgila.ga tgcgaacctt tgcatacctti caaccagttg ttcgacaact ^r '£

7 ü

AG OJTTGGC AA GGTTTGGC CT TGTTATCTg Ia AGCT GTTTTG AGAGGTCAAG TCAAACCfcTT CCAAACCGGA ACAATAGACT TC (3ACAAAAC TCTCCAGTTC

10

11, H

CCO JTGTT GGT TMCTCTTCT CAACCATGGG jAACCA TTGCA ATTGCACGTC GGAESUICCA ATTGAGAAGA GTTGGTACCC TTGGTiAACGT TAACGTGCAG

^r IA

, 15 BgIII BamEl

GAO JAAAGC Cg TCTCTGGTTT GAGATCTG

CTATTTqGGC AGAGACCAAA CTCTAGACCTA G ^r 16

Table XIX SCEPOSECTIONSOLIGONUCLEOTIDES

1. GATCCAGATCTTTGACTACTTTGTT

2. TCTCAACAAAGTAGTCAAAGATCTG

3. GAGAGCTTTGGGTGCTCAAAAGGAAG

4. ATGGCTTCCTTTTGAGCACCCAAAGC

5. CCATTTCCCCACCAGACGCTGCTT

6. GCAGAAGCAGCGTCTGGTGGGGAA

7. CTGCCGCTCCATTGAGAACCATC

8. CAGTGATGGTTCTCAATGGAGCG

9. ACTGCTGATACCTTCAGAAAGTT

10. GAATAACTTTCTGAAG GTATCAG

11. ATTCAGAGTTTACTCCAACTTCT

12. CTCAAGAAGTTGGAGTAAACTCT

13. TGAGAGGTAAATTGAAGTTGTACAC

14. ACCGGTGTACAACTTCAATTTACCT

15. CGGTGAAGCCTGTAGAACTGGT

16. CTGTCACCAGTTCTACAGGCTTC

17. GACAGATAAGCCCGACTGATAA

18. GTTGTTATCAGTCGGGCTTAT

19. CAACAGTGTAGATGTAACAAAG

20. TCGACTTTGTTACATCTACACT

Table XX SCEPO SECTION 3

BamHI BgIII 1,

CAGATCTTTG ACTACTTTGT TjGAGAGCTTT GTCTAGAAAC TGAtGAAACA ACTCφGAAA 2

GGGTGCTCAA AAGGAAC JCCA T TCCCUACC AGACGCTGCT T CTGC CGCTC

cccACGAGTT ttccttcggt Iaaggggtgg tctgcgacga agacggcgag 4 '£

7 9

cattgagaac catcJactgct gataccttca gaaagtt Ltt ca gagtttac

GTAACTCTTG GTAGTGAC1GA CTATGGAAGT CTTTCAATAA GfECTCAAATG 8 10 ' ^f

13

TCCAACTTCT ItGAGA GGTAA ATTGAAGTTG TACACJCGGTG AAGCCTGTAG AGGTTGAAGA ACTCJTCCATT TAACTTCAAC ATGTGGCCijC TTCGGACATC

14. 16 _

17

YES.

AACTGG JGAC AGA TiUGCCC GACTGATAis b AAC AGTGTAG TTGACCACTG TCJTATTCGGG CTGACTATTG TTqTCACATC

18

Sai l

ATGTAACAAA G TACATTGTTT CAGCT 20

Table XXI SCEPO GEN

-1 +1

HindIII ArgAla

AGCTTGGATA AAAGAGCTCC ACCAAGATTG ATCTGTGACT CGAGAGTTTT

ACCTAT TTTCTCGAGG TGGTTCTAAC TAGACACTGA GCTCTCAAA

GGAAAGATAC TTGTTGGAAG CTAAAGAAGC TGAAAACATC ACCACTGGTT

CCTTTCTATG AACAACCTTC GATTTCTTCG ACTTTTGTAG TGGTGACCAA

GTGCTGAACA CTGTTCTTTG AACGAAAACA TTACGGTACC AGACACCAAG

CACGACTTGT GACAAGAAAC TTGCTTTTGT AATGCCATGG TCTGTGGTTC

GTTAACTTCT ACGCTTGGAA ACGTATGGAA GTTGGTCAAC AAGCTGTTGA

CAATTGAAGA TGCGAACCTT TGCATACCTT CAACCAGTTG TTCGACAACT

AGTTTGGCAA GGTTTGGCCT TGTTATCTGA AGCTGTTTTG AGAGGTCAAG

TCAAACCGTT CCAAACCGGA ACAATAGACT TCGACAAAAC TCTCCAGTTC

CCTTGTTGGT TAACTCTTCT CAACCATGGG AACCATTGCA ATTGCACGTC

GGAACAACCA ATTGAGAAGA GTTGGTACCC TTGGTAACGT TAACGTGCAG

GATAAAGCCG TCTCTGGTTT GAGATCTTTG ACTACTTTGT TGAGAGCTTT

CTATTTCGGC AGAGACCAAA CTCTAGAAAC TGATGAAACA ACTCTCGAAA

GGGTGCTCAA AAGGAAGCCA TTTCCCCACC AGACGCTGCT TCTGCCGCTC

CCCACGAGTT TTCCTTCGGT AAAGGGGTGG TCTGCGACGA AGACGGCGAG

CATTGAGAAC CATCACTGCT GATACCTTCA GAAAGTTATT CAGAGTTTAC

GTAACTCTTG GTAGTGACGA CTATGGAAGT CTTTCAATAA GTCTCAAATG

Table XXI (continued)

-1 + 1

Hindll Arg Al a

TCCAACTTCT TGAGAGGTAA ATTGAAGTTG TACACCGGTG AAGCCTGTAG AGGTTGAAGA ACTCTCCATT TAACTTCAAC ATGTGGCCAC TTCGGACATC

AACTGGTGAC AGATAAGCCC GACTGATAAC AACAGTGTAG

TTGACCACTG TCTATTCGGG CTGACTATTG TTGTCACATC "* '

Sail

ATGTAACAAA G TACATTGTTT CAGCT

The pooled SCEPO sections were sequenced in M13 and sections 1.2 and 3 were isolatable from the phage as Hind III / KpnI, KpnI / BglII and BglII / Sal! Fragments.

The presently most preferred expression system for SCEPO gene products is a scretion system based on S. cerevisiae α-factor secretion, as described in copending U.S. Patent Application No. 487753, filed April 22, 1983, by Grant A. Bitter, published 31 October 1984 as European Patent Application 0123294. Briefly, the system includes constructions in which DNA encoding the leader sequence of the yeast a-factor gene product is located immediately 5 'to the coding region of the exogenous gene to be expressed. As a result, the translated gene product contains a leader or signal sequence that is "processed off" by an endogenous yeast enzyme in the course of secretion of the remaining product It was not necessary to incorporate such a codon at the -1 position of the SCEPO gene As can be seen from Table XXI, the alanine (+1) coding sequence precedes a linker sequence which is a direct insertion into a Plasmid allowed containing the DNA for the first 80 residues of the α-factor leader sequence following the α-factor promoter The particularly preferred construction for SCEPO gene expression involves four-fold ligation, including the above-mentioned SCEPO-Section fragment and fragment of HindIII / SalI degradation of the plasmid paC3 From the resulting paC3 / SCEPO, the a-factor promoter and the leader sequence and isolated the SCEPO gene by digestion with Bam HI and ligated into the Barn HI-degraded plasmid pYE to arrive at the expression plasmid pYE / SCEPO.

It should be apparent upon consideration of the examples that numerous extraordinarily valuable products and procedures have been produced by the present invention in its many aspects.

In another aspect of the present invention, the cloned DNA sequences described herein encoding human and monkey EPO polypeptides are remarkably valuable for the information contained therein regarding the amino acid sequence of mammalian erythropoietin that have not been obtained to date. apart from parts of analytical procedures of isolates of naturally occurring products. The DNA sequences are also remarkably valuable as products that can be used to extensively describe the microbial synthesis of erythropoietin by a variety of recombinant techniques. On the other hand, the DNA sequences provided by the present invention are useful for the generation of novel and beneficial viral and circular plasmid DNA vectors, novel and beneficial transformed and transferred microbial procaryotic and eucaryotic host cells (including bacterial and yeast cells, and mammalian cells grown in culture, as well as novel ones and beneficial methods of cultivated growth of such microbial host cells capable of expressing EPO and EPO products.

The DNA sequences of the present invention are also remarkably suitable materials for use as labeled probes in the isolation of EPO and related protein-encoding cDNA and genomic DNA sequences from mammals other than the human and monkey species specifically discussed herein. The content to which the DNA sequences according to the invention are used in the different alternative method for the synthesis of Prokin (eg in insect cells) or in the genetic therapy of humans or other mammals can not yet be calculated. It is expected that the DNA sequences of the present invention may be useful in the development of transgenic mammalian species which may serve as eucaryotic "hosts" for the production of erythropoietin and erythropoietin products in amounts See, generally, Polmiter et al., Science, 222 (4625) , 809-814 (1983).

From this point of view, it is clearly not intended that the illustrative examples limit the scope of the present invention, and numerous modifications and variations will be apparent to those skilled in the art. As an example, while to the DNA sequences pertaining to the illustrative examples, as this application provides amino acid sequence information essential to the preparation of the DNA sequence, the invention also encompasses those DNA sequences produced as recited in the wake of the art the EPO amino acid sequences can be constructed. These may encode EPO (as in Example 12), as well as EPO fragments and kEPO polypeptide analogues (ie, "EPO products") having one or more of the biological properties of naturally occurring EPO, but not others (or others in different degrees).

The DNA sequences disclosed by the present invention thus include all DNA sequences suitable for safe expression of a polypeptide product in a procaryotic or eucaryotic host cell, which product comprises at least a portion of the primary structural arrangement and one or more of the biological properties of Having erythropoietin selected from:

(a) the DNA sequences listed in Tables V and VI;

(b) DNA sequences which hybridize to those listed under (a) or fragments thereof; and

(c) DNA sequences which hybridize, but with degeneracy of the genetic code, to the DNA sequences defined under (a) and (b). In this regard, it is noteworthy, for example, that existing allelic monkey and human EPO gene sequences and the gene sequences of other mammalian species are expected to hybridize to sequences of Tables V and VI or to fragments thereof. In addition were also - albeit under degeneration of the genetic

Codes - the SCEPO and ECEPO genes and the prepared or mutated cDNA or genomic DNA sequences encoding different EPO fragments and analogs also hybridize to the above DNA sequences. Such hybridizations could easily be performed under the hybridization conditions described herein with regard to the initial isolation of monkey and human EPO-encoding DNA or more severe conditions to reduce background hybridization if desired.

Improved hybridization methodologies of the invention, illustratively applied to the above DNA / DNA hybridization screens, are also applicable to RNA / RNA and RNA / DNΑ screening. Mixed probe techniques, as discussed herein, generally consist of a number of improvements in hybridization techniques that allow for faster and more reliable polynucleotide isolation. These many individual process improvements include:

improved colony transfer and maintenance methods; Use of nylin-based filters, such as Gene-Screen and Gene-Screen-Plus, to allow re-probing with the same filtering and repeated use of the filters. Application of New Protease Treatments [eg, Taubetal., Anal. Biochem., 126, p.222-230 (1982)]; Using very low single concentrations (in the range of 0.025 picomoles) of a larger number of mixing probes (eg the numbers above 32); and conducting hybridization and post-hybridization steps under severe temperatures close to (ie within 4 ⁰ C and preferably within 2 ° C of) the lowest calculated dissociation temperature of any of the mixed probes employed. The improvements together lead to results that were unexpected from the outset. This is further emphasized by the fact that mixed probing methods with four times the number of probes reported hitherto as successfully employed in the same cDNA screens of messenger RNA species of relatively small amount have succeeded in isolating a single gene in one Gene library were applied under screenings of 1500000 phage plaques. This was achieved in substantial contrast to the view published by Anderson et al., Supra, that mixing probe screening methods are incapable of isolating mammalian protein genes if the corresponding RNA's are not available. The features disclosed in the foregoing description, in the following claims and / or in the accompanying drawings, both separately and in combination, may be the basis for the realization of the invention in its various forms.

Claims (4)

A method of determining a specific single-stranded polynucleotide of unknown sequence in a heterogeneous cellular or viral sample including multiple single-stranded polynucleotides, characterized in that "(a) a mixture of labeled single-stranded polynucleotide probes with uniformly varying sequences of the bases is prepared, each of the probes being potentially of sequence is complementary to bases, which is the allegedly only once existing copy to the polynucleotide to be determined; (b) fixing the sample on a solid substrate; (c) treating the substrate with the sample fixed thereon to attenuate further binding of polynucleotides thereto, except by hybridization to polynucleotides in the sample; (d) contacting the treated substrate with the sample fixed thereon in a volatile manner with said mixture of labeled probes under conditions which only facilitate hybridization between fully complementary polynucelotides; and (e) the specific polynucleotide is determined by observing the occurrence of a hybridization reaction between it and a fully complementary probe from the mixture of labeled probes, as compared to the appearance of a higher density of the labeled material on the substrate at the site of the specific polynucleotide to a background density of labeled material resulting from non-specific binding of labeled probes to the substrate, the improvement being that the use of 32 mixing probes is in excess and one or more of the following features occurs. (1) use of a nylon-based paper as a solid substrate; (2) treatment with a protease in step (c); (3) use of individually labeled probe concentrations of approximately 0.025 picomole; and (4) Use as one of the hybridization conditions in step (d), severe temperatures close to. 4 ° C from the lowest calculated Td of any of the probes used.