BG60442B2 - Dna encoding csf-1 and accompanying recombinant systems - Google Patents

Abstract

Colony stimulating factor CSF-1 (lymphokin) is useful in surmounting immunosuppression induced from heamotherapy, or as a result of other causes. CSF-1 protein is produced in usable quantities by recombinant methods, including cloning and expression of mice and human DNA sequences encoding it.

Description

TECHNICAL FIELD

The invention relates to the use of recombinant technology for the production of lymphokines, usually produced at low concentrations. More specifically, the invention relates to the cloning and expression of DNA sequences encoding factor-1 for stimulating human colonies (CSF-1).

BACKGROUND OF THE INVENTION

The ability of some factors produced at low concentrations in a large number of tissues to stimulate the growth and development of bone marrow precursors in granulocytes and / or macrophages is known. The presence of such factors in sera, urine samples and tissue extracts of a large number of species has been demonstrated experimentally in vitro by measuring the stimulation of colony formation by bone marrow cells seeded in semi-solid media. There are no known in vitro studies. Because the factors induce the formation of such colonies, they are called Colony Stimulating Factors (CSF).

It has been proven that there are at least four subclasses of human CSF proteins that can be defined according to the type of cells found in the colonies formed. One subclass, CSF-1, results in the formation of colonies containing predominantly macrophages. Other subclasses produce colonies containing neutrophilic granulocytes and macrophages, or predominantly neutrophilic granulocytes or neutrophilic and eosinophilic granulocytes and macrophages.

There are murine factors similar to the first three of the listed human CSFs. In addition, murine factor IL-3 induces colonies of murine bone marrow cells containing all these cell types plus megakaryocytes and cells in a wide variety of combinations. These CSFs have been reviewed by Dexter, T.M., Nature, / 1984/309: 746 and Vadas, M.A. et al. Immunol / 1983/130: 793.

The invention relates to a recombinant method for the production of proteins belonging to the first of said CSF-1 subclasses. This subclass is characterized by radioimmune and radioreceptor experiments, for example antibodies in the presence of purified CSF-1 are able to specifically suppress CSF-1 activity without affecting the biological activity of the other subclasses, and macrophage J744 cell lines contain receptors that specifically bind CSF-1 as described in Das, S.K. et al., Blood (1981) 58: 630.

Methods for purification of various CSF proteins have been published and are described in the following literature. Stanley, E.R. J. Biol Chem / 1977/252: 4305 reports the purification of CSF proteins from murine L929 cell line with a specific activity of about 1 x 10 'units / mg, which also stimulate the production of macrophages predominantly. Wejid, A. et al., Blood / 1982/60: 238, describes purification of murine CSF-1 L-cells to overt homogeneity using a rabbit antibody column and reports the first 25 amino acids of the murine sequence / Ben-Avery, C. M. et al., PrI Natl Acad Sci (USA) (1985/882: 448).

Stanley, E.R. et al., J. Biol Chem / 1977/252: 4305-4312 Discloses a Method for Purification of CSF-1 from Human Urine and Das, S.K. et al., Blood / 1981/58: 630; J. Biol Chem / 1982/257: 13679 receives human CSF-1 from urine with a specific activity of 5x10 'units / mg, which produces only colonies of macrophages and outlines the glycosylation relationship of CSF-1 proteins prepared from cultured murine L-cells and of human urine, with their activities. Wang, F.F. et al., J. Cell Biochem / 1983/21: 263 isolated human CSF-1 from urine with a specific activity of 10 * units / mg. Weihid A. et al. detected purification of human urinary CSF-1 with a specific activity of 0.7-2.3 x 10 'units / mg on a rabbit antibody column (Exp Hemat / 1094/12: 434).

V.M. et al., J. Biol Chem / 1979/254: 6226 reported the production of CSF protein from human carcinoma culture / MIAPaCa / cells that grew from murine granulocytes and macrophage clones. The resulting protein has a specific activity of about 7x10 '/ mg.

Partially purified preparations of various CSFs have been described as isolated from the conditioned medium from murine lung cells (Foggio, S.C. et al., Biochemistry / 1978/17: 3109; Burgus, A.V. et al., J. Biol Chem (1977/252: 1998 as isolated from human T-limb foblast cells / Lusis, A.J. et al., Blood / 1981/57: 13; US Patent 4,438,032) as conditioned medium isolated from human placenta in an obvious homogeneity and specific activity 7 x 10 'units / mg (View, M. et al. Biochemistry / 1980/19: 3846).

A significant difficulty for any useful application of CSF proteins in general and CSF-1 in particular is the lack of distinctive and characterized forms in amounts sufficient to enable their practical therapeutic application. The invention overcomes the difficulties of supplying human and mouse CSF-1 in sufficient quantities by recombinant techniques.

CSF protein from a different subclass, mouse and human GM-CSF, was purified and the cDNA $ was cloned. This protein has been shown to be different from CSF, in particular CSF-1, Gough, et al, Nature / 1984/309: 763-767. Mouse IL-3 is cloned by Fung, M.C. et al., Nature / 1984/307: 233, and also by Yokota, T. et al. PNAS / 1984/81: 1070-1074; Wong,

G.G.et al, Science / 1985/228: 810-815; Lee, F., et al. PNAS / 1985/82: 4360-4364; and Cantrell M.A.et al, PNAS / 1985/82: 6250-6254.

SUMMARY OF THE INVENTION

The invention relates to recombinant CSF-1 protein, including biologically active proteins containing modifications of the native amino acid sequence of the native protein. CSF-1 protein in recombinant form can be quantified, predominantly modified by regulation of the posttranslational process provided by the host or deliberately modified at the gene or protein level to enhance the desired properties. Such activity, for example, is possessed by muteins with deletions of substantial portions of the carboxy terminal one-third of the polypeptide. The availability of CSF-1 in the recombinant form affords mobility and quantitative advantages that make the therapeutic use of the protein impossible, which is impossible with the native protein.

On the other hand, the invention relates to an isolated ADH sequence encoding recombinant CSF-1, to recombinant expression systems of this sequence, to vectors containing them, to recombinant hosts transformed with these vectors, and to recombinant-producing cultures protein. The invention also relates to methods of producing recombinant protein and materials important for production.

The invention also relates to compositions containing CSF-1, which are pharmaceutically and therapeutically useful, and to the administration of these compositions.

BRIEF DESCRIPTION OF THE DIAGRAMS

FIG. 1 shows the partial amino acid sequences of CSF-1 from human urine or mouse L-929 cells as determined from purified native proteins.

FIG. 2 shows the sequence of some oligomers from murine CSF-1.

Fig. 3 shows the sequence of an oligomer used to produce the human genomic CSF-1.

FIG. 4 shows a sequenced portion of

3.5 kb Hind III fragment encoding human CSF-1 sequences, and derived amino acid sequences for exon regions.

Figure 5 shows the DNA sequences and derived amino acid sequences for the NK clone encoding CSF-1.

Figure 6 shows a comparison of the activities of CSF-1 and other colony stimulating factors in enhancing the ability of macrophages to kill tumor cells.

FIG. 7 shows the results of fractionation of MIAPaCa RNA into a glucose gradient.

Method of carrying out the invention

A. Definitions "Colony stimulating factor-1 / CSF1 /" refers to a protein exhibiting diverse activity as described in the CSF-1 level. For example, when applied to a standard in vitro colony stimulating sample according to Metcalf, D., J. Cell Physiol / 1970/76: 89, primary macrophage colonies are formed. Native CSF-1 is a glycosylated dimer. Dimerization may be necessary for the activity. According to the invention and by definition, CSF-1 are both dimeric and monomeric forms. The monomeric form can be converted to dimeric by providing in vitro, on intracellular conditions, and the monomer is useful as an antigen in the production of anti-CSF-1 antibodies.

Specific specificity has also been established: human CSF-1 operates on both human and mouse bone marrow cells. Therefore, "human" CSF-1 should be positive for a specific mouse radioreceptor sample according to Das, S.K., Et al, Blood / 1981/58: 630, although there is not necessarily a complete correlation. Protein biological activity is generally inhibited by neutralized human urine CSF-1 antisera (Das, S.K.et, al Supra). However, in certain special circumstances (for example, a specific antibody preparation recognizes the CSF-1 epitope not only for biological purposes, which epitope is not present in the tested CSF-1 mutein.) This criterion may be absent.

Some other properties of CSF-1 have been discovered more recently, including the ability of this protein to stimulate the secretion of E-series prostaglandins, interleukin-1, and interferon by mature macrophages / Moore, R., et al, Science / 1984/223 : 178. The mechanism for these late-discovered abilities has not yet been discovered and, for definition purposes, the definition of feasibility of the definition rests on the ability to stimulate the formation of monocytes / colonies by macrophages derived from bone marrow cells of a suitable type as starting material, in most cases (see above), the inhibition of this activity by neutralizing the antiserum against purified human urine CSF-1 treated for the specific species gave a positive response in radioreceptor experiments. / It is known that the proliferative effect of CSF-1 is restricted to cells from the mononuclear phagocyte lineage (Stanely, ER, The Lymphokines / 1981 /, Stewart, WE, II et al, Humana Press, Clifton, NJ /, p. 102 -132 / and CSF-1 receptors are restricted to these cell lines (Byrne, PV, et al, Cell Biol / 1981/91: 848).

As with all proteins, the exact chemical structure depends on a number of factors. When ionizing amino groups and carboxy groups are present in a molecule, a particular protein can be obtained as an acidic or basic salt or in a neutral form. All those preparations which retain their activity when ch is placed in a suitable medium are included in the definition. Further, the primary amino acid sequence can be increased by branching, using sugar residues (glycosylation) or with other additional molecules such as lipids, phosphates, acetyl groups, and more, most commonly by binding to saccharides. The primary amino acid structure can be aggregated to form complexes, most commonly dimers. Native human CSF-1 is isolated as a highly glycosylated dimer. Some aspects of this increase are accomplished through post-translational host production systems, and other such modifications can be made in vitro. In any case, such modifications are included in the definition insofar as the activity of the protein as defined is not degraded. It is anticipated that such modifications may cause quantitative or qualitative changes in activity either by enhancing or reducing protein activity in various experiments.

Individual amino acid residues from the chain can be modified by oxidation, reduction or other derivatives, and the protein can be cleaved to produce fragments that retain activity. Such modifications that do not disrupt activity do not displace the protein sequence from the definition.

Modifications of the primary structure itself by deletion, addition, or substitution of amino acids included in the sequence during translation can be effected without destroying the activity of the protein. Such substitutions or other alterations result in proteins having an acid sequence that fall within the definition of proteins "having an amino acid sequence substantially equivalent to that of CSF-1". In fact, the human and the derivative CSF-1 proteins do not have identical but similar primary amino acid sequences, indicating a high degree of homology.

For convenience, the amino acid sequence of the mature protein from the monomer moiety of the dimer protein shown in Figure 5, derived from the cloned NNA, illustrated herein, is designated _m CSF-1 (mature CSF-1). Figure 5 shows the presence of a signal sequence of 32 residues that is most likely cleaved by the secreting action of mammalian cells, " CSF-1 is represented by 1-224 amino acids shown in this figure. Specifically included in the definition of human CSF-1 are muteins whose monomers and dimers are ca _m CSF-1 and linked to CSF-1 forms, indicated by their differences from _B CSF-1. A derivative of CSF-1 of other species may be adapted to the definition of "human" _B CSF-1 by virtue of its manifestation in the desired activity direction, as specified in relation to the human substrate.

For convenience, the amino acid sequence of _B CSF-1 will also be used as reference and other sequences substantially equivalent to CSF-1 activity will be indicated with reference to the sequence shown in FIG. The replacement of an amino acid will be recorded with respect to the amino acid residue that is substituted. For example, ser _M CSF1 refers to a protein having the sequence shown in Figure 5, except that the 90-position amino acid is a series instead of cysteine. Deletions are indicated by a sign followed by the number of amino acids omitted from the N-terminus or by the number of amino acids remaining when the residues are deleted from the C-terminus when the number is followed by a minus sign. Thus, CSF-1 refers to CSF-1 of Figure 5, where the first 4 amino acids of the N-terminus are deleted; refers to CSF-1 where the last 94 amino acids following the amino acid at the 130 position have been dropped. In the following, an example is given in which ^ jCSF-1 contains an aspartate residue encoded by the gene (FIG. 5/) at position 59 instead of a tyrosine residue encoded by J ^ HK, h ^ jj-CSF-I, which only contains amino acids 1-158 of _m CSF-1.

"Operationally connected" refers to an adjacent location where the normal operation of the components can be improved. The coding sequences "operatively linked" to control sequences refer to a configuration where the coding sequence can be expressed under the control of those sequences.

The term "control sequences" is from seed to DNA sequences required for the expression of an operably linked coding sequence in a single host organism. Control sequences suitable for prokaryotes, for example, a promoter, possibly an operator sequence, a ribosome binding site, and possibly other poorly understood sequences. Eukaryotic cells are known to use promoters, polyadenylation signals, and enhancers.

An "expression system" refers to sequences containing the desired coding sequence and operably linked control sequences so that the transformed host with these sequences is able to produce the encoded protein. In order to effect the transformation, the expression system may be incorporated into a vector. In addition, the relevant DNA can also be integrated into the host chromosome.

The terms "cell", "cell line" and "cell culture" are interchangeable and any such designation includes their offspring. "Transformants" or "transformed cells" include the primary cells of the subject and the cultures derived from them regardless of the number of transfers. It is also understood that the offspring may not be exactly identical in terms of DNA content due to directional or spontaneous mutations. The mutant generation, which has the same functionality as the screened ones, is included in the primary transformed cells. When special designations are provided, this becomes clear from the context.

C. General description

The CSF-1 proteins of the invention are capable of both stimulating the production of bone marrow precursor monocyte macrophage precursors and enhancing the efficiency of the immune system and stimulating such functions in differentiated cells as lymphokine secretion in mature macrophages. On the one hand, these proteins are useful as an aid in chemotherapy. Chemotherapy treatment involves suppression of the immune system, but often despite the successful destruction of the tumor cells targeted, this treatment results in the death of the subject due to the side effect of the chemotoxic agents on the cells of the immune system. Administration of CSF-1 to such patients restimulates the immune system due to the ability of CSF-1 to mediate and enhance the growth and differentiation of bone marrow precursor derived cells in macrophages and monocytes and to stimulate some of the functions of these mature cells, in order to prevent side effects and prevent patients from becoming prone to secondary infection. Other patients who would benefit from this treatment, including those who have been treated for leukemia through bone marrow transplants, are often in an immune-suppressed state to avoid rejection. In these cases, immunosuppression may also be reversible by administration of CSF-1.

Typically, any subject suffering from immunosuppression due to chemotherapy, bone marrow transplantation, or other incidental forms of immunosuppression such as a disease (such as spin) could benefit from the CSF-1's ability to find pharmaceutical use. Subjects may also be administered an additional amount of pre-differentiated macrophages to assist the innate systems, the macrophages being produced in vitro by bone marrow cell cultures or suitable CSF-1 treated preparations. Such preparations include those monocytes from the blood of patients that may be cultured in this manner and returned for local or systemic therapy.

The ability of CSF-1 to stimulate the production of lymphokines by macrophages and enhance their ability to kill target cells also makes it directly applicable in the treatment of neoplasms and infections.

CSF-1 stimulates the production of interferons from murine macrophage derivatives (Fleit, NV, etyal, J.Cell Phsiol (1981/108: 347) and human, partially purified CSF-1 from MIAPaCa cells stimulate poly (1). : poly / C / -induced production of interferon and TNF by human monocytes, as illustrated subsequently. In addition, CSF-1 stimulates the production of myeloid CSF by human blood monocytes.

Also shown below is the ability of murine CSF-1 (from L-conditioning-cell medium) to stimulate normal CH3 / HeN murine peritoneal macrophages to kill murine TU5 sarcoma target cells. This activity is more effective when CSF-1 is used for pre-treatment and during the effective phase. The ability of CSF-1 to act in this way is significantly greater than that indicated for other colony stimulating factors shown in Fig. 6. In addition, the ability of mouse cells to attack viruses is enhanced by CSF-1.

Mouse CSF-1 is known to stimulate mouse macrophages to exert cytostatic action on P815 tumor cells / Wing, EJ, et al, J. Clin Invest (1982/69: 270) or not to kill target leukemic cells / Ralph, P. et al, Cell Immunol (1983/76: 10).

B Nogawa, R.T., et al, Cell Immunol / 1980/53: 116 has described that CSF-1 can stimulate macrophages to digest and kill yeast.

In addition, to overcome immunosuppression per se, CSF-1 can be used to destroy invading organisms and malignant cells indirectly by stimulating macrophage secretion and activity.

CSF-1 according to the invention can be administered in a conventional, standard manner for the administration of protein substances. Injectable administration is preferable, the forms being solutions, suspensions, emulsions or solid compositions for injection. Suitable excipients may be, for example, Ringer's solution, Hank's solution, water, saline, glycerol, dextrose solutions and the like. In addition, CSF-1 according to the invention can be pre-incubated with a cell preparation in order to stimulate appropriate responses. Also, the whole preparation or supernatant can be introduced into the site. As shown, products produced in response to the stimulation of CSF-1 by different types of blood cells are effective against the desired targets and the properties of the blood cells themselves to invade invading viruses and neoplasms can be enhanced. The cells of the subject itself can be separated and used in the same way, or for example monocytes or lymphocytes from another compatible individual, to be used in incubation.

Despite the existence of a pattern of activity of said known CSF-1, the responsible protein has never been produced in sufficient pure form and in a quantity that allows the sequence to be determined, neither in sufficient pure form and amount to provide either protein or coding DNA. were available, it was not possible to optimize the structural modifications by offering such alternatives as those mentioned in point A and using the protein with its therapeutic nature.

The present invention eliminates these defects. Through a large number of additional purification methods, purified CSF1 is obtained from human urine to provide a known amino acid sequence, thereby allowing the construction of an oligomeric DNA template. Samples are needed to obtain the coding sequence of the entire protein. The approach illustrated below uses certain probes with respect to a human N-terminal sequence from a human genomic bank to obtain suitable segments of coding sequences. The human genomic cloned sequence can be directly expressed using its own control sequences or by constructing suitable mammalian systems capable of reproducing intones. Genomic sequences can also be used to probe human DNA, a bank derived from cSF-Ι-producing cell lines, to produce cDNA encoding this protein. Properly prepared DNA can be directly expressed in COS or CV-1 cells and can be constructed into vectors suitable for expression in a wide variety of hosts.

Thus, the complete human CSF-1 coding sequence can be obtained from which expression vectors applicable to a large number of host systems can be constructed and the coding sequence can be expressed. The large number of hosts with vectors suitable for these sensitivities, further suggested, make it possible to choose between post-translational reproduction systems and environmental factors for conformational regulation of the protein thus obtained.

C. Suitable hosts, control systems and methods

The production of the recombinant form of CSF-1 includes the following. First, a DNA encoding the mature protein (used as including all muteins / preproteins or fusion of CSF-1 protein with an additional sequence that does not disrupt its activity, or with an additional sequence that can be cleaved under controlled conditions) is obtained. peptidase / to produce the active protein. If the sequence is not interrupted by introns, it is suitable for expression in any host. If entrones are present, expression is obtained in mammals or other eukaryotic systems capable of reproducing them. The sequence must be in a form that is convenient to cut and recover. The cut or restored coding sequence is then operatively linked to suitable control sequences in a replicating expression vector. The vector is used to transform a suitable host cultured under suitable conditions to produce recombinant CSF-1. Optionally, CSF1 can be isolated from the medium or from the cells, in some cases it is not necessary to separate and purify the protein when some impurities are allowed. For example, in vitro culturing of cells from which a lymphokine factor will be isolated for administration to a subject does not require absolute purity. Direct use in patient therapy will require purification of the protein produced.

Each of the preceding steps can be accomplished in different ways. For example, the desired coding sequences can be obtained by preparing appropriate DNA from cellular RNA and manipulating the DNA in an appropriate manner to obtain the entire sequence. Alternatively, genomic fragments can be obtained and used directly with a suitable host. The construction of expression vectors operating in a large number of hosts is accomplished by the use of suitable replicons and control sequences, as will be seen below. In the absence of suitable restriction sites, they can be added at the end of the coding sequence to provide an excised gene for inversion into these vectors.

The control sequences, expression vectors and transformation methods depend on the type of host cells used for gene expression. Typically, prokaryotic, yeast, and mammalian cells are suitable as hosts. Once native CSF-1 is secreted as a glycosylated dimer, hosts with systems capable of their own posttranslational processes are preferred. Although prokaryotic hosts are usually the most efficient and most suitable for protein production, eukaryotic cells, in particular mammalian cells, are preferred because of their processing capabilities. Bacterial recombinant CSF-1 requires in vitro dimerization. In addition, it is more certain that the native signal sequence will be recognized by mammalian host cells, which secures secretion and facilitates purification.

C.1. Control sequences and corresponding hosts.

Most commonly, prokaryotes are represented by different E. coli strains. Other strains of microorganisms such as bacilli, such as Bacillus subtilis, different species of Pseudomonas, or other bacterial strains, may also be used. Such prokaryotic systems utilize plasmid vectors containing replication sites and control sequences derived from host compatible species. For example, E. coli is typically transformed by derivatives of the pBR 322 plasmid derived from E. coli strain-Bolivar, et al, Gene / 1977 / 2:95. pBR322 contains genes for ampicillin and tetracycline resistance and provides additional markers that can either be retained or destroyed in the design of the desired vector. Commonly used prokaryotic control sequences, defined as containing promoters for transcription initiation, optionally with a ribosomal binding sequence operator, include commonly used promoters such as lactamase / penicillazan / lactose / promoter systems / Chang, et al, Nature / 1977/198: 1056 / and tryptophan / trp / promoter system / Goeddelt, et al, Nucleisc

Acids Res (1980/8: 4057) and a lambda derivative P _L promoter and N-gene ribosome binding site (Shimatakre, et al, Nature / 1981/292: 128), which is applicable as a portable control cassette, as claimed in US Pat. 4 711 845. However, any available prokaryotic compatible promoter system may be used.

In addition to bacteria and eukaryotic organisms such as yeast, they can also be used as a host. Most commonly used laboratory strains of Sacchgaromyces cerevisiale or yeast, despite the large number of commonly available strains. Vectors using the two-micron replication origin described in Broach, JR, Meth Enz / 1983/101: 307, other plasmid vectors suitable for expression in yeast, are known in Stinchcomb, et al, Nature / 1979/282: 39, Tschempe, et al, Gene / 1980/10: 157 and Glarke, L, et al, Meth Enz / 1983/101: 300. Control sequences for yeast vectors include promoters for the glycolytic enzyme system / Hess.et al, J. Adv Enzyme Reg / 1968/7: 149; Holland, et, Biochemistry, (1978/17: 4900). Additional promoters known in the art include the 3-phosphoglycerate kinase promoter (Hitzeman, et al, J. Biol Chem (1980/255: 2073), and others for glycolytic enzymes such as glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruxase, decarboxylase, decarvatase, decarvatose 6-phosphatisomerase,

3-phosphoglyceramutase, pyruvate kinase, triosophosphatisomer phosphoglucose isomerase and glucokinase. Other promoters that have the added benefits of growth-controlled transcription are promoter regions of alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degrading enzymes related to nitrogen metabolism, and enzymes responsible for the use of maltose and galactose. / Holland, ibid /. Terminator sequences are thought to be desired at the 3'-end of the coding sequence. Such terminators are detected on 3'-untranslated regions following the coding sequences of yeast derivatives. Many of the illustrated vectors contain control sequences derived from plasmid repo 46 containing the enolase gene / Holland, MJ, et al J. Biol chem / 1981/256: 1385 / or the LEU ₂ gene derived from ΥΕ _ρ 13 / Broach, J ., et al, Gene (1978).

8: 121 /, although any vector containing a yeast compatible promoter, start of replication, and other control sequences is appropriate.

It is also possible to express genes encoding polypeptides in host cells in cultures derived from multicellular organisms. See, for example, Tissue Culture, Academic Pfress, Grusz and Patterson, editors / 1973 /. Useful host cell lines include murine myeloma N 51, VERO and HeLa cells and Chinese hamster ovary cells. / CHO /. Expression vectors for such cells typically include promoters and control sequences compatible with mammalian cells, for example the commonly used late and early promoter of Simon virus 40 (SV40) / Fiers, et al, Nature (1978/273: 113) or other viral promoters such as those derived from polyoma, Adenovirus 2, bovine papilloma virus or sarcoma virus, or immunoglobulin promoters and promoters. General aspects of mammalian host cell transformation systems are described by Axel, patent US 4 399 216. Also, "enhancer" regions are important in optimizing expression - these are usually sequences located above the promoter regions. Replication origins can be obtained when viruses are needed. However, integration into the chromosome is a common mechanism in DNA replication and eukaryotes. Plant cells are also now able to be used as hosts and control sequences compatible with plant cells, such as the nopalin synthetase promoter and polyadenylation signaling systems (Depicker, A., et al, J. Mol Appl Gen (1982/1: 561). are available.

C.2. Transformations

Depending on the host cells used, transformation is performed using standard techniques suitable for these cells. Calcium treatment using calcium chloride, as described by Cohen, S.N., Proc Natl Sci / USA / 1972/69: 2110, is used for prokaryotes and other cells containing significant cellular barriers. Agrobacterium tumefaciens infection has been used in some plant cells (Shaw, SN, et al, Gene (1983/23: 315). For mammalian cells without such cell walls, the calcium phosphate precipitation method according to Graham and van der Eb, Virology / 1978/52: 546 is preferred. Yeast transformation is accomplished by the method of van Solingen, R., et al, J. Bact / 1977/130: 946 and Hsiao, C. L. et al, Proc Natl Acad Sei / CAIH / 1979/76: 3829.

B.3. Northern blot mRNA testing, DNA testing or genomic testing

RNA was fractionated for "Northern blot" by thin-layer agarose gel electrophoresis under absolutely denaturing conditions using formaldehyde / Maniatis, T., et al, Molecular Cloning (1982 / Cold Spring Harbor Press, pp 202-203) or 10 mM methylmercury (CH ₃ HgOH) / Bailey, JM, et al, Anal Biochem (1976) 70: 75-85; and Sehgal, PB, et al, Nature (1980/288: 95-97) as a denaturing agent. For methylmercury gels, 1.5% gels were prepared by mixing with working buffer (100 mM boric acid, 10 mM sodium sulfate, 1 mM EDTA, pH 8.2), cooled to 60 ° C and added 1 / 100 volume fraction 1M CH ₃ HgOH. The RNA was dissolved in 0.5 x working buffer and denatured by incubation in 10 mM methylmercury for 10 min at room temperature. Glycerol (20%) and bromophenol blue (0.05%) were added to fill the samples. Samples were electrophoresed at 500-600 wolt-hr with recirculation of the buffer. After electrophoresis, the nude was washed for 40 min with 10 mM 2-mercaptoethanol to remove methylmercury and spots prepared by transferring the RNA from the gel to a membrane filter.

DNA or genomic banks are screened using a column or disk hybridization procedure. Bacterial colonies or phage discs are placed on double nitrocellulose filter paper (S&S type BA-85). Disks or colonies were lysed and DNA was fixed by treating the sequences for 5 min with 500 mM NaOH, 1.5 M NaCl. The filters were washed twice for 5 min each time with 5 x standard salt citrate (SSC), air-dried and baked for 2 h at 80 ° C.

Gels for spot or double DNA filters or genomic screening are subjected to re-hybridization at 25 ° -42 ° C for 6-8hrs10mL filter DNA hybridization buffer without assay / 0-50% formamide, 5-6x SSC, pH 7.0, 5x Denhard / polyvinyl-pyrrolidine solution plus Ficoli and bovine serum albumin; 1 x 0.02% of each, 2050 mM sodium phosphate buffer with pH 7.0, 0.2% SDS, 20 μg / ml poly U / DNA test, and 50 μg / ml denatured salmon sperm DNA /. The samples were then hybridized by incubation at a suitable temperature for about 24 to 36 hours using a hybridization buffer containing a kinase sample (for oligorems). Longer DNA or genomic fragments are designated by nick-translation or primary extension.

The conditions of pre-hybridization and hybridization depend on the desired binding and vary, for example, according to the length of the sample. Typical conditions for relatively long samples (e.g., more than 30 to 50 nucleotides) are the use of a temperature of 42 to 55 ° C and a hybridization buffer containing 20% to 50% formamide. At the lower degree of binding required for oligomeric samples of about 15 nucleotides, lower temperatures of 25 ° to 42 ° C and lower concentrations of formamide from 0% to 20% are used. For longer samples, the filters may be washed, for example, four times for 30 min each time at 40 ° C-55 ° C, with 2 x SSC, 0.2% SDS and 50 mM sodium phosphate buffer at pH 7. They can then be washed twice with 0.2% x SSC and 0.2% SDS, air-dried and subjected to autoradiography at -70 ° C for two to three days. The shorter samples are washed under milder conditions.

C.4. Designing vectors.

When constructing suitable vectors, the desired coding and control sequences utilize standard linkage and restriction techniques, which are clarified in the art. Isolated plasmids, DNA sequences or systemic oligonucleotides are cleaved, reshaped and reconnected in the desired form.

Claims (33)

Claims An isolated DNA sequence encoding a human CSF-1 polypeptide or a murine CSF-1 polypeptide, wherein the isolated polypeptide stimulates the formation of primary macrophage colonies in an in vitro CSF-1 assay. The DNA sequence of claim 1 encoding human CSF-1. The DNA sequence of claim 2, wherein the human CSF-1 has an amino acid sequence substantially equivalent to that of the native human CSF-1. The recombinant DNA sequence of claim 2, wherein the amino acid sequence of human CSF-1 is linked to that of mCSF-1 by deletion or conservative substitution of one or more amino acids located between positions 150 and 224, including mCSF-1 , as shown in Figure 5. The DNA sequence of claim 2, wherein the human CSF-1 has an amino acid sequence that is related to that of mCSF-1, by deletion or conservative substitution of one or more amino acids at positions 51 and 52 and / or positions 191,192 and 193 from mCSF-1 shown in FIG. The DNA sequence of claim 2, wherein the human CSF-1 has an amino acid sequence linked to that of mCSF-1 by deletion or conservative substitution of one or more amino acids at positions 15-20 and / or positions 75-84 of mCSF-1 as shown in Figure 5. The DNA sequence of claim 2, wherein the human CSF-1 has an amino acid sequence that is linked to that of mCSF1 by deletion or substitution of the tyrosine residue at position 59 of CSF-1. The DNA sequence of claim 2, wherein the human CSF-1 has an amino acid sequence that is selected from the group consisting of mCSF-1, _Jg -CSF-1, _J0 -CSF-1, gln _J2 CSF-1 and asp _J9 CSF-1. The DNA sequence of claim 1, wherein CSF-1 has the amino acid sequence of native mouse CSF-1. The DNA sequence of claim 2, which encodes a protein with an amino acid sequence encoded in pHCSF-1α or in CSF-17. A replicative cloning vector comprising the DNA sequence encoding a human CSF-1 polypeptide or a murine CSF-1 polypeptide that stimulates colony formation of primary macrophages in an in vitro CSF1 assay, and an operative replicon in a unicellular organism. The vector of claim 11, wherein the coding CSF-1 sequence encodes a protein with an amino acid sequence substantially equivalent to that of human mCSF-1. 13. The vector of claim 12, wherein the encoding CSF-1 sequence encodes a protein having an amino acid sequence selected by the group consisting of _{mCSF-1, Vf ja CSF-1,5 £ o} - CSF- 1, gin _j2 CSF-l * asp ₃ , CSF-1. The vector of claim 11, wherein the coding CSF-1 sequence encodes a protein with an amino acid sequence encoded in pH SF-1α or pcCSF-17. An expression system comprising the DNA of claim 1 operatively linked to suitable control sequences. The expression system of claim 15, wherein the recombinant DNA sequence encodes a human CSF-1 having an amino acid sequence substantially equivalent to that of human mCSF-1. 17. The expression system of claim 15, wherein the recombinant DNA sequence encodes human CSF-1 with an amino acid sequence selected by the group consisting of mCSF-Ι, ^ _jg CSF-1 CSF-1, gin _J2 CSF-1 and asp _J9 CSF-l. The expression system of claim 15, wherein the recombinant DNA sequence encoding CSF-1 includes pHCSF1a or pcCSF-17. The expression system of claim 15, which is housed in a vector that can replicate in suitable host cells. 20. An expression system according to claim 16, arranged in a vector, k3 that can replicate in suitable host cells. 21. Recombinant host cells transformed with an expression system according to claim 15. 22. Recombinant host cells transformed with an expression system according to claim 16. A method of producing recombinant CSF-1, which comprises culturing the cells of claim 21 under conditions effective for the production of said CSF-1. A method of producing recombinant CSF-1, which comprises culturing the cells of claim 22 under conditions effective for the production of said CSF-1. 25. The DNA of claim 2, cloned into pHCSF-1α. 26. The DNA of claim 2, cloned into pcCSF-17. 27. An isolated DNA sequence encoding a human CSF-1 polypeptide, wherein CSF-1 has an N-terminal amino acid sequence comprising Glu-Glu-Val-Ser-Glu-Tyr-Cys-Ser-HisMet-Ile-Gly-Ser-Gly-His-Leu-Gln-Ser-Leu-GlnArg-Leu-Ile-Asp-Ser-Gln-Met- Glu-Thr-Ser-CysGln-Ile-Thr-Phe-Glu-Phe-Val-Asp-Gln-Glu-GlnLeu, and wherein said polypeptide stimulates colony formation of primary macrophages in an in vitro CSF-1 assay. An isolated DNA sequence encoding a murine CSF-1 polypeptide, wherein CSF-1 has an N-terminal amino acid sequence including Lys-Glu-Val-Ser-Glu-His-Cys-Ser-HisMet-Ile-Gly-ASn-Gly -His-Leu-Lys-Val-Leu-GlnGln-Leu-Ile-Asp-Ser-Gln-Met-Glu-Thr-Ser-, and wherein said polypeptide stimulates colony formation of primary macrophages in an in vitro CSF-1 assay. A replicative cloning vector comprising the DNA sequence of claim 27 or 28, and an operative single-cell organism replicon. An expression system comprising a DNA sequence according to claim 27 or 28, operatively linked to appropriate control sequences. 31. Recombinant host cells transformed with an expression system comprising a DNA sequence according to claim 27 or 28, operatively linked to appropriate control sequences. 32. A method of producing recombinant CSF-1, which comprises culturing the recombinant host cells transformed with an expression system comprising the DNA sequence of claim 23 operatively linked to suitable control sequences under conditions effective for the production of said CSF-1. 33. A method of producing recombinant CSF-1, which comprises culturing recombinant host cells transformed with an expression system comprising the DNA sequence of claim 28, operatively linked to suitable control sequences under conditions effective for the production of said CSF-1.