WO1986000089A1 - Eukarygotic hybrid vectors and preparation of polypeptides - Google Patents

Abstract

Extrachromosomal ribosomal DNA (rDNA) can be used as a vector for introducing a foreign gene into eukaryotic cells. Eukaryotic hybrid vectors comprising an extrachromosomal rDNA or functional fragments thereof and a foreign structural gene coding for a polypeptide, and methods for their preparation, characterized in that a foreign structural gene coding for a polypeptide and optionally additional DNA sequences are introduced in vitro or in vivo into an extrachromosomal rDNA or functional fragments thereof. A eukaryotic cell transformed with a hybrid vector and methods for the preparation thereof, characterize in that a transformable eukaryotic cell is treated with the eukaryotic hybrid vector under transforming conditions.

Description

EUKARYGOTIC HYBRID VECTORS ANDPREPARATION OF POLYPEPTIDES

Field of the Invention

The invention pertains to the field of recombinant DNA technology and concerns a method for the preparation of polypeptides with the aid of genetically engineered eukaryotic cells, said genetically engineered cells, novel vectors useful for the transformation of eukaryotic cells and methods for the preparation of said eukaryotic cells and said vectors, and the use of extrachromosomal ribosomal DNA as vectors.

Background of the Invention

The art of manipulating procaryotic and eukaryotic cells in such a manner that they express a polypeptide which is normally not or not at the same level expressed by these cells has made great progress in recent years. The material, necessary in recombinant DNA technology comprises a structural gene coding for the desired polypeptide, a vector into which said gene can be inserted, a host which receives said vector with said gene and which upon proliferation reproduces said vector with said gene and produces the desired polypeptide, and a number of enzymes. Genes coding for polypeptides, such as for human insulin, growth hormone, interferons, tissue plasminogen activator and the like, have been used. Commonly used vectors are especially plasmids (extrachromosomal genetic elements consisting of a circular duplex of DNA which can replicate independently of chromosomal DNA), and further bacteriophages (phages; bacterial viruses composed of a linear duplex of DNA surrounded by a protein wall) and cosmids (plasmid DNA packed into a phage particle). The hosts used are prokaryotic (without a nucleus) or eukaryotic (with a nucleus) cells, for example prokaryotic organisms, such as bacteria, e.g. Escherichia coli, or eukaryotic yeasts, such as Saccharomyces cerevisiae, or plant, animal or human cells. Important enzymes are DNA polymerase (for converting single-stranded DNA to the double-stranded form) , reverse transcriptase (for synthesizing complementary DNA upon messanger RNA templates), RNA polymerase (for the preparation of a radioactive RNA copy of a double-stranded DNA), terminal transferase (for addition of oligodeoxynucleotide tails to 3'-ends of DNA duplex), DNA ligase (for sealing single-strand nicks in DNA duplexes and for covalently linking flush-ended DNA duplexes), restriction endonucleases (for cleavage of DNA duplexes at defined sequences), DNase I (for very limited treatment of double-stranded DNA so as to introduce nicks), nucleases ( e.g. SI, for destroying single-stranded nucleic acid), exonucleases (for removing nucleotides from the ends of duplex DNA), polynucleotide kinase (for labelling 5'-ends of polynucleotides by transfer of γ- ³²P from ATP) and poly A polymerase (for polymerisation of AMP from ATP onto free 3'-hydroxyl group of RNA).

The major steps in recombinant DNA technique comprise

a) the preparation of the structural gene coding for the desired peptide, which gene can be prepared by reverse transcription from the corresponding mRNA, from the whole DNA by shot-gun cloning, or by chemical synthesis,

b) the incorporation of the obtained gene into an appropriate vector by means of various enzymes mentioned above,

c) the transfer of the newly created vector carrying the gene into a recipient host (transformation), d) the selection of the transformed host from the untransformed hosts, usually by culturing under such conditions where only the transformed host survives,

e) the culturing of the selected transformed host under conditions to allow expression of the foreign structural gene, and, if required,

f) the isolation and purification of the expressed polypeptide.

Each of the major steps consists of one or more single steps, and a number of variations are known for performing these steps. For the successful preparation and finally isolation and purification of a desired polypeptide by the recombinant DNA technology each step of the multistep synthesis has to be carefully planed and performed. Although the general concepts are known, a number of unforeseeable pitfalls and drawbacks may prevent reaching the desired goal. Difficulties are mainly due to the unforeseeable behavior of the living cells, when they are forced to accept the vector with the foreign structural gene, transcribe it to the corresponding mRNAs, and translate the latter to the desired polypeptide.

Just one of the drawbacks is that the cells have the tendency to get rid of the foreign elements. In order to select the transformed from the untransformed cells, and to avoid prevailing of the latter during culturing, the vector with the foreign structural gene must contain an expressible DNA coding for a phenotypical trait allowing for survival of the transformed cell under conditions whereby the untransformed cells cannot survive. Such selective marker DNA for bacteries are for example providing for resistance against ampicillin or against tetracycline, and for yeasts, such as Saccharomyces cerevisiae, the genes providing survival in an auxotrophic genetic background, for example LEU2 or HIS3. Any novel means or methods, or improvements thereof, improving one or more steps within the multistep synthesis will be appreciated by the skilled worker in this art, because such alternatives provide the possibility of improving the entire synthesis. Thus, it would be highly desirable to have a vector which does not need a selective marker DNA, and which also could be used for the transformation of further cells which could not be transformed with the vectors hitherto known.

Parts of extrachromosomal ribosomal DNA (rDNA) have been used in yeast vector construction (35). However, entire extrachromosomal rDNA molecules have not been used as vectors up to now. Such extrachromosomal DNA elements carry the genes coding for ribosomal RNA. They are known to exist in a number of lower eucaryotes, for example in the acellular slime mold Physarum polycephalum (P.p.) [ for reviews see Braun, R. and Seebeck, T. (1), and Long, E. O. and David, (2)]. The extrachromosomal rDNA molecules replicate independently from the chromosomes and are present throughout the mitotic life cycle of the organism. They remain stabely associated with the cell and its nucleolus, where they are contained almost exclusively during the interphase.

Autonomous replication of small pieces of DNA has often been achieved in lower organisms, such as yeasts. The stability of such sequences, however, is usually poor. In yeast, for example, in the absence of a selective pressure, such sequences, as a rule, are lost within 20 to 40 generations. The stability of such DNA molecules can be improved when sequences originating from a centromere are linked thereto. However, such centromere sequences are expected to have no stability properties in heterologous systems.

Accordingly, it was unlikely that extrachromosomal elements would be stabely inherited in a heterologous cell. Surprisingly it was found that extrachromosomal rDNA is useful as a vector. It was found to have a high transformation rate in eukaryotic cells, including plant cells, and to remain stably associated during proliferation of the cells.

Object of the Invention

It is an object of the present invention to use extrachromosomal rDNA as a vector for introducing a foreign gene into eukaryotic cells, to provide novel vectors composed of an extrachromosomal rDNA and a foreign structural gene coding for a polypeptide, which novel vectors surprisingly remain stabely associated with eukaryotic cells, also with heterologous eukaryotic cells, transformed therewith, and which are not lost during cell replication.

As the transformation rate is very high and the vector is not lost during cell replication, it is not necessary to provide the vector with a selective marker DNA.

It is a further object of the invention to provide eukaryotic cells transformed with the novel vectors.

Further objects of the invention are methods for the preparation of said vectors and said transformed eukaryotic cells, and their use in methods for the preparation of polypeptides.

Terma and Abreviations used in the Description and Claims Eukaryotic hybrid vector: a DNA by means of which a DNA coding for a polypeptide (insert) can be introduced into a eukaryotic cell.

Extrachromosomal ribosomal DNA (rDNA); a DNA found in unicellular eukaryotes outside the chromosomes, carrying one or more genes coding for ribosomal RNA and replicating autonomously (independent of the replication of the chromosomes) . Palindromic DNA: a DNA molecule with one or more centers of symmetry.

DNA: desoxyribonucleic acid.

rDNA: ribosomal DNA.

RNA: ribonucleic acid.

rRNA: ribosomal RNA.

Insert; a DNA sequence foreign to the rDNA, consisting of a structural gene and optionally additional DNA sequences.

Structural gene: a gene coding for a polypeptide and being equiped with a suitable promoter, termination sequence and optionally other regulatory DNA sequences, and having a correct reading frame.

Promoter: a recognition site on a DNA strand to which RNA polymerase binds to initiate transcription.

Signal sequence: a DNA sequence coding for an amino acid sequence attached to the polypeptide which binds the polypeptide to the endoplasmatic reticulum and is essential for protein secretion. (Selective) Genetic marker; a DNA sequence coding for a phεnotypical trait by means of which transformed cells can be selected from untransformed cells.

Detailed Description of the Invention

1. Eukaryotic Hybrid Vectors

The present invention concerns eukaryotic hybrid vectors comprising an extrachromosomal ribosomal DNA (rDNA) or functional fragments thereof and an insert containing a foreign structural gene coding for a polypeptide and optionally additional DNA sequences. The present invention concerns further the use of extrachromosomal ribosomal DNA or functional fragments thereof as vectors for introducing a foreign gene into eukaryotic cells.

Extrachromosomal rDNAs useful in the construction of the present hybrid vectors are known and can be derived from various unicellular eukaryotes, especially from protozoa of the two phyla Sacoromastigophera and Ciliophera, such as of the orders Dictyosteliida, Physarida, Tetrahymenina, Penlculina and Sporodotrichina, for example of Dictyostelium dlscoideum, Tetrahymena species, Paramecium apecies, Oxytricha species, Stylorichia species, and especially Physarum polycephalum (for review see 1 and 2). The sources of extrachromosomal rDNA are readily available (3).

The extrachromosomal rDNA is a palindromic or sometimes also nonpalindrotoic molecule which can be circular, however is mostly linear. One or more copies may be present in a single eukaryotic cell nucleus depending on whether it is a micronucleus ("genetic" nucleus) or a macronucleus ("metabolic" nucleus). The transcription units for the rRNA may be interrupted. The extrachromosomal rDNA is isolated from its source by conventional methods, such as equilibrium density centrifugation, for example with cesium chloride (CsCl).

Functional fragments of the extrachromosomal rDNA are such DNA segments which have retained a high transformation efficacy without the necessity of selection, stability and/or replication functions. Such fragments can be obtained by various restriction enzymes, such as Bgl II, Bam HI, Taq I, and the like, and optionally subsequent separation, e.g. by agarose gel electrophoresis. The restriction map of the extrachromosomal rDNA of Physarum polycephalum is disclosed in reference (31).

The foreign structural gene of the insert is a DNA coding for a wide variety of polypeptides, including glycosylated polypeptides, such as enzymes which can be used, for example, for the production of nutrients and for performing enzymatic reactions in chemistry, or non-enzymatic polypeptides, for example hormones, polypeptides with immunomodulatory, anti-viral and anti-tumor properties, antibodies, viral antigens, vaccines, clotting factors, foodstuffs and the like. Examples of such polypeptides are insulin, growth hormones, lymphokines, anti-renin antibodies, soraatostatin and, in particular, interferons, such as human interferon of the α-, β- or γ-type, e.g. human leukocyte interferon, human fibroblast interferon, human lymphoblastoid interferon, or human immune interferon, Hepatitis surface antigen, and the like. As an example the structural gene is coding for human tissue plasminogen activator (TPA).

The structural gene for transformation of plant cells provides for example for resistance against plant pests, excess temperatures, or dryness, or improves growth of the whole plant or parts thereof.

The structural genes, may be linked to additional DNA sequences, preferably to a promoter and/or other regulatory sequences which should be selected depending on the recipient cell.

For example, if the recipient cell is a yeast, preferably the promoter of a highly expressed yeast gene is used, for example of Saccharomyces cerevisiae. Thus, the promoters of the TRP1 gene, the ADHI or ADHII gene, acid phosphatase (PH03 or PH05) gene, isocytochrome gene or a promoter involved with the glycolytic pathway, such as the promoter of the enolase, glyceraldehyde-3phosphate dehydrogenase (GAPDH), 3-phosphoglycerate kinase (PGK), hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase and glucokinase genes, can be used. Preferred vectors of the present invention contain promoters with transcriptional control, e.g. the promoters of the PH05, ADH II and GAPDH genes, which can be turned on or off by variation of the growth conditions. For example, the PH05 promoter can be repressed or derepressed solely by increasing or decreasing the concentration of inorganic phosphate in the medium. Promoters of mammalian cells are for example virus promotors, such as HTLV, SV40, vaccinia promoter and the like. Plant promoters are for example those of the Ti plasmids or the cauliflower mosaic virus.

The promoter is operably linked to the coding region of the structural gene so as to ensure effective expression of the mRNA and then the polypeptide.

Other sequences optionally comprised by the inserts of the present hybrid vectors are for example signal sequences, such as one of those naturally linked to a promoter, e.g. the PH05 signal sequence, or to the polypeptide, e.g. the TPA signal sequence, or the signal sequence of a yeast invertase or α-factor gene. Alternatively, fused signal sequences may be constructed by ligating part of the signal sequence of the gene naturally linked to the promoter used with part of the polypeptide signal sequence.

The hybrid vectors according to the invention may contain additional DNA sequence(s) which are inessential or less important for the function of the promoter, but which may perform other important functions, for example, in the propagation of the cells transformed with said hybrid vectors. Such additional DNA sequence(s) may be derived from prokaryotic and/or eukaryotic cells and may include chromosomal and/or extra-chromosomal DNA sequences. For example, the additional DNA sequences may stem from (or consist of) plasmid DNA, such as prokaryotic or eukaryotic plasmid DNA, viral DNA and/or chromosomal DNA, such as bacterial, yeast or higher eukaryotic chromosomal DNA, especially from bacterial plasmids, preferably Escherichia coli plasmid pBR322 or related plasmids, bacteriophage λ, yeast 2μ plasmid, and/or yeast chromosomal DNA.

The additional DNA sequences may carry a replication origin and/or a selective genetic marker. Optionally a genetic marker can be used which facilitates the selection for transformants due to the phenotypic expression of the marker. Suitable markers are particularly those expressing antibiotic resistance or, in the case of auxotrophic mutants, genes which complement host lesions. Corresponding genes confer, for example, resistance to the antibiotic cycloheximide or provide for prototrophy in an auxotrophic mutant.

In view of the palindromic character of the extrachromosomal rDNA the hybrid vectors of the present invention may contain one or more, especially two, inserts, which are inserted to the right and/or to the left of the center(s) of symmetry. The reading direction of the inserts in the rDNA can be from outside to inside or vice versa. The restriction sites divide the extrachromosomal rDNA into three or more segments. For example, there are two Bglll, two Hindlll, two EcoRI, and two BamHI restriction sites on each site of the center of symmetry of the rDNA of Physarum polycephalum which accordingly can be divided into three to five segments. The inserts are located between the two segments formed by the restriction sites, preferably at the restriction sites further away from the center of symmetry.

The eukaryotic hybrid vectors of the present invention are prepared by conventional methods, in that the insert containing the structural gene and optionally additional DNA-sequences, is introduced into an extrachromosomal rDNA or a functional fragment thereof.

An in vitro method consists in cutting the rDNA by a suitable restriction enzyme into segments and joining them with the inserts, if necessary after attaching appropriate linkers and/or preparing the ends of the segments and the inserts in another conventional method for successful ligation.

Various techniques are used to join DNAs in vitro. Blunt ends (fully base-paired DNA duplexes) produced by certain restriction endonucleases may be directly ligated with T4 DNA ligase. More usually, DNAs are linked through their single-stranded cohesive ends and covalently closed by a DNA ligase, e.g. T4 DNA ligase. Such singlestranded "cohesive termini" may be formed by cleaving the DNA with endonucleases producing staggered ends (the two strands of the DNA duplex are cleaved at different points at a distance of a few nucleotides). Single strands can also be formed by the addition of nucleotides to blunt ends or staggered ends using terminal transferase ("homopolymeric tailing") or by simply digesting back one strand of a blunt-ended DNA with a suitable exonuclease, such as λ-exonuclease. A further approach to the production of staggered ends consists in ligating to the blunt-ended DNA a chemically synthesized linker DNA which contains a recognition site for a staggered-end forming endonuclease and digesting the resulting DNA with the respective endonuclease.

In order to be efficiently expressed, the structural gene must be properly located with respect to sequences containing transcriptional (promoter) and translational functions (ribosome binding sites). Furthermore, the construction of the eukaryotic hybrid vector should be done in such a way that it allows correct transcription initiation and termination.

The ligation mixture containing the desired eukaryotic hybrid vector is used in the transformation step or after enrichment of the desired vector by gel electrophoresis.

The rDNA segments obtained after treatment with the restriction nuclease may be used as a mixture in the ligation experiment with the insert DNA, or alternatively, after chromatographic separation into the single segments, whereby a stepwise ligation is possible. In the latter case, for example, after digestion of the rDNA with e.g. Bglll, the large DNA segment and the two smaller DNA segments are separated, whereupon the inserts are first ligated to the large segment followed by ligation of the small segments. Alternatively it is also possible to ligate the inserts first to the small segments and in a second step to the large segment of the rDNA. The introduction of the inserts into the extrachromosomal rDNA can also be performed in vivo, namely in cells, e.g. bacteria, animal, plant or especially yeast cells, transformed with the entire rDNA. For this purpose the cells, e.g. Saccharomyces cerevisiae, are transformed with whole rDNA in a manner described below, whereupon the transformed cells are transformed with the insert DNA containing the structural foreign gene, optionally the controlling promoter and fermination sequences, and flanking rDNA sequences from the rDNA restriction sites. The transformation with the whole rDNA and the conditioned inserts may also be carried out simultaneously.

After transformation both rDNA and insert sequence are joined by homologous recombination within the cell.

In order to facilitate detection of the transformants the cotransformation technique may be applied, whereby e.g. the cotransforming plasmid Yep 13 may be used (32).

After screening and selection of cells containing the desired hybrid vector, the latter may be isolated and used for transforming other cells, or transferred directly by cell-cell fusion.

2. Transformation of the eukaryotic cells with the eukaryotic hybrid vectors

Another aspect of the present invention involves a process for the production of transformed eukaryotic cells capable of producing a polypeptide, which process comprises transforming a eukaryotic host with a eukaryotic hybrid vector described in chapter 1.

Any eukaryotic cells can be used as recipients for this hybrid vector. Examples are eukaryotic fungi, such as yeasts, Aspergillus sp., Neurospora sp., Podospora sp., Penicillium sp., Cephalosporium sp., Mucor sp., especially Saccharomyces cerevisiae, Schizosaccharomyces pombe, Aspergillus niger, Neurospora crassa, Podospera anserina, Penicillium chrysogenum or Cephalosporium acremonium. The hybrid vectors of the invention are also suitable for the transformation of all plants, especially those of the systematic groups Angiospermae and Gymnospermae.

Among the Gymnospermae, the plants of the Coniferae class are of particular interest.

Among the Angiospermae, plants of particular interest are, in addition to deciduous trees and shrubs, plants of the following families: Solanaceae, Cruciferae, Compositae, Liliaceae, Vitaceae, Chenopodiaceae, Rutaceae, Bromeliaceae, Rubiaceae, Theaceae, Musaceae or Gramineae and of the order Leguminosae, in particular of the family Papilionaceae. Preferred plants are representatives of the Solanaceae, Cruciferae and Gramineae families.

To be particularly mentioned are plants of the species Nicotiana, Petunia, Hyoscyamus, Brassica und Lolium, as for example, Nicotiana tabacum, Nicotiana plumbagenifolia, Petunia hybrida, Hyoscyamus muticus, Brassica napus, Brassica rapa and Lolium multiflorum.

In the field of transformation of plant cells, interest focuses in particular on the high yield cultivated plants such as maize, rice, wheat, barley, rye, oats and millet.

All plants which can be produced by regeneration from protoplasts can also be transformed with the hybrid vectors of this invention. So far it has not been possible to manipulate genetically representatives of the Gramineae family (grasses), which also comprises cereals. It can be shown that graminaceous cells, including cereal cells, can be transformed with the present hybrid vector. In the same way, transformation of cultivated plants of the genus Solanum, Nicotiana, Brassica, Beta, Pisum, Phaseolus, Glycine, Helianthus, Allium, wheat, barley, oat, Setaria, rape, rice, Cydonia, Pyrus, Malus, Rubus, Fragaria, Prunus, Arachis, Secale, Panicum, Saccharum, Coffea, Camellia, Musa, Ananas, Vitis or Citrus is possible and desirable, even if the total yields and crop areas are smaller worldwide.

Of the Cruciferae the genus Brassica is especially mentioned, for example rape seed, black and white mustard, as well as cabbage and beets, for example Brassica rapa especially Brassica rapa cv Just Right.

Further examples of eukaryotic cells are derived from invertebrates, especially insects, such as Drosophila melanogaster, and from vertebrates especially mammalian cells, for example, HeLa cells, Chinese Hamster Ovary cells, T- and B-cell hybridomas, Melanoma cells, e.g. Bowes melanoma, and the like.

The transformation of the eukaryotic cells with the eukaryotic hybrid vector is performed by methods known in the art.

The invention concerns also a method for producing transformed eukaryotic cells according to the invention, characeterized in that a transformable eukaryotic cell is treated with the eukaryotic hybrid vector under transforming conditions.

In particular, the present method is characterized in that,

a) for the transformation of a eukaryotic cell having a cell wall, the cell wall is removed and the obtained cell wall free spheroplast or protoplast is treated with the eukaryotic hybrid vector, e.g. in the presence of polyethyleneglycol (PEG) and Ca²⁺. ions, or

b) for the transformation of eukaryotic cells without a cell wall, the eukaryotic cell is treated with the eukaryotic hybrid vector by calcium phosphate precipitation of the vector DNA onto the recipient cells, and, if necessary, regenerating the cell wall and selecting of or screening for the transformed cells.

Various enzymes are known to digest the cell walls of fungi and plant cells and to prepare thereby transformable cells. Preferred are glucosidases, such as snail gut juices (e.g. Glusulase^® or Helicase^®) or enzyme mixture obtained from microorganisms (e.g. Zymolyase^®), which are used in osmotically stabilized solutions (e.g. 1M sorbitol).

The transformation of cell wall free cells and the spheroplasts (fungi) or protoplasts (plants) with the eukaryotic hybrid vector according to the invention can be accomplished with the entire ligation mixture containing the vector or after enrichment of the vector, and is carried out by the general methods known in the art, for yeasts for example as described by Hinnen et al., Proc. Natl. Acad. Sci. USA, 75, 1929 (1978),

Whereas protoplasts, cell culture cells, cells in plant tissues, pollen, pollen tubes, egg-cells, embryo-sacs or zygotes amd embryos in different stages of development are representative examples of plant cells which are suitable starting materials for a transformation, protoplasts are preferred on account of the possibility of using them direct without further pretreatments.

Isolated plant protoplasts, cells or tissues can be obtained by methods which are known per se or by methods analogous to known ones.

Isolated plant protoplasts which are also suitable starting materials for obtaining isolated cells and tissues can be obtained from any parts of the plant, for example from leaves, embryos, stems, blossoms, roots or pollen. It is preferred to use leaf protoplasts. The isolated protoplasts can also be obtained from cell cultures. Methods of isolating protoplasts are described e.g. in Gamborg, O.L. and Wetter, L.R., Plant Tissue Culture Methods, 1975, 11-21. The transfer of the new hybrid vectors into plant cells is effected direct without using a natural system for infecting plants such as a plant bacterium, a plant virus, or transfer by insects or phytopathogenic fungi. This is achieved by treating the plant cells which it is desired to transform direct with the hybrid vector to be transferred by introducing the vector and plant protoplasts into a suitable solution and leaving them therein until the vector has been taken up by the protoplasts. The transformation frequency can be increased by combining this step with techniques which are employed in microbiological research for gene transfer, for example by treatment with poly-L-ornithine or poly-L-lysine, liposome fusion, DNA protein complexing, altering the charge at the protoplast membrane, fusion with microbial protoplasts, or calcium phosphate co-precipitation, heat shock or electroporation, and, in particular, by treatment with polyethylene glycol, heat shock and electroporation. Also a combination of the last three mentioned techniques can be used.

Suitable solutions into which the hybrid vector and the receptor protoplasts are introduced are preferably the osmotically stabilised culture media employed for protoplast cultures. Numerous culture media are already available which differ in their individual components or groups of components. However, the composition of all media is in accordance with the following principle: they contain a group of inorganic ions in the concentration range from about 10 mg/ℓ to several hundred mg/ℓ (so-called macroelements such as nitrate, phosphate, sulfate, potassium, magnesium, iron), a further group of inorganic ions in maximum concentrations of several mg/ℓ (so-called microelements such as cobalt, zinc, copper, manganese), then a number of vitamins (for example inositol, folic acid, thiamine), a source of energy and carbon, for example saccharose or glucose, and also growth regulators in the form of natural or synthetic phytohormones of the auxin and cytokinin classes in a concentration range from 0.01 to 10 mg/ℓ. The culture media are additionally stabilised osmotically with sugar alcohols (for example mannitol) or sugar (for example glucose) or salt ions (for example CaCl₂), and are adjusted to a pH in the range from 5.6 to 6.5.

A more detailed description of conventional culture media will be found, for example, in Koblitz, H., Methodische Aspekte der Zellund Gewebezuchtung bei Gramineen unter besonderer Berϋcksichtigung der Getreide, Kulturpflanze XXII, 1974, 93-157.

A particularly suitable technique of transformation for yeast spheroplasts and plant cell protoplasts is "polyethylene glycol treatment", where the term "polyethylene glycol" within the scope of this invention denotes not only the substance polyethylene glycol itself, but will also be understood as generic term for all substances that likewise modify the protoplast membrane and are employed e.g. in the field of cell fusion. The term thus also comprises other polyhydric alcohols of longer chain length, for example polypropylene glycol (425 to 4000 g/raole), polyvinyl alcohol or polyhydric alcohols whose hydroxyl groups are partially or completely etherified, as well as the detergents which are commonly employed in agriculture and tolerated by plants, and which are described e.g. in the following publications: "Mc Cutcheon's Detergents and Emulsifiers Annual", MC Publishing Corp., Ridgewood New Jersy, 1981; Stache, H. , "Tensid-Taachenbuch", Carl Hanser Verlag, Munich/Vienna, 1981.

If polyethylene glycol itself is used, then it is preferred to use a polyethylene glycol having a molecular weight in the range from 1000 to 10,000 g/mole, preferably from 3000 to 8000 g/mole.

Of the above mentioned substances, it is preferred to use polyethylene glycol itself.

In the polyethylene glycol treatment, the procedure can be for example such that either a suspension of the protoplasts is added to a culture medium and then the vector is added in a mixture of polyethylene glycol and culture medium, or, advantageously, protoplasts and vector are first added to the culture medium and then polyethylene glycol is added.

With the hybrid vectors of the present invention a surprisingly high and reproducible transformation frequency is achieved by means of the techniques described above. However, this frequency may be even greatly improved on by the appropriate techniques described in more detail hereinafter.

In the process of this invention, electroporation and heat shock treatment may also be advantageous techniques.

In electroporation [Neumann, E. et al., The EMBO Journal 7, 841-845 (1982)], protoplasts are transferred to an osmoticum, for example a mannitol/magnesium solution and the protoplast suspension is introduced into the electroporator chamber between two electrodes. By discharging a condenser over the suspension, the protoplasts are subjected to an electrical impulse of high voltage and brief duration, thereby effecting polarisation of the protoplast membrane and opening of the pores in the membrane.

In the heat treatment, protoplasts are suspended in an osmoticum, for example a solution of mannitol/calcium chloride, and the suspension is heated in small containers, for example centrifuge tubes, preferably in a water bath. The duration of heating will depend on the chosen temperature. In general, the values are in the range of 40°C for 1 hour and 80°C for 1 second. Optimum results are obtained at a temperature of 45°C over 5 minutes. The suspension is subsequently cooled to room temperature or lower.

It has also been found that the transformation frequency can be increased by inactivating the extracellular nucleases. Such an inactivation can be effected by using divalent cations that are tolerated by plants, for example magnesium or calcium, and also preferably by carrying out the transformation at a high pH value, with the optimum pH range being from 9 to 10.5.

Surprisingly, the use of the present hybrid vectors results in the enormous increase in transformation frequency that has long been an objective in the field of genetic engineering. Furthermore, the use of selection markers is unnecessary.

After the transformation, the cell walls have to be regenerated. This regeneration is conveniently done by embedding the spheroplasts into agar. For example, molten agar (about 50°C) is mixed with the spheroplasts. Upon cooling the solution to growth temperatures (about 30°C) a solid layer is obtained. This agar layer is to prevent rapid diffusion and loss of essential macromolecules from the spheroplasts or protoplasts and thereby facilitates regeneration of the cell wall. However, cell wall regeneration may also be obtained (although at lower efficiency) by plating the spheroplasts onto the surface of preformed agar layers.

Optionally, if a selection marker is present, the regeneration agar is prepared in a way to allow regeneration and selection of the transformed cell at the same time. If genes coding for enzymes of amino acid biosynthetic pathways are used as selective markers, the regeneration is preferably performed in a minimal medium agar. If very high efficiencies of regeneration are required following a two step procedure is advantageous: (1) regeneration of the cell wall in a rich complex medium, and (2) selection of the transformed cells by replica plating the cell layer onto selective agar plates.

Screening methods include in situ hybridization with a labeled DNA fragment homologous to sequences of the hybrid vector [e.g. according to Hinnen et al. (30)], in situ immunoassays, provided that the antibody of the product of the introduced gene is available, or other screening methods which measure gene products encoded by the transforming vector. Alternatively, the eukaryotic cell can be co-transformed with a hybrid vector according to the invention and a second vector containing a genetic marker.

The invention also relates to eukaryotic cells transformed with eukaryotic hybrid vectors comprising an extrachromosomal rDNA and a structural gene.

The transformed eukaryotic cells of the present invention have the advantage of growing without selection pressure. Optionally a selective pressure can be applied, especially for obtaining a pure transformed cell line.

3. Cultivation of the transformed eukaryotic cells and isolation of the expressed polypeptide

The invention concerns further a method for the preparation of a polypeptide, characterized in that eukaryotic cells according to the present invention transformed with a eukaryotic hybrid vector according to the present invention coding for said polypeptide are cultured, and, when required, said polypeptide is isolated.

The transformed eukaryotic cells according to the present invention are cultured by methods known in the art in a liquid medium containing assimilable sources of carbon, nitrogen, inorganic salts, and if necessary growth promoting substances.

Various carbon sources are usable. Examples of preferred carbon sources are assimilable carbohydrates, such as glucose, maltose, mannitol or lactose, or an acetate, which can be used either alone or in suitable mixtures. Suitable nitrogen sources include, for example, amino acids, such as Casamino acids, peptides and proteins and their degradation products, such as tryptone, peptone or meat extracts, furthermore yeast extract, malt extract, corn steep liquor, as well as ammonium salts, such as ammonium chloride, sulphate or nitrate, which can be used either alone or in suitable mixtures. Inorganic salts which may be used include for example sulphates, chlorides, phosphates and carbonates of sodium, potassium, magnesium and calcium. Additionally, the nutrient medium may also contain growth promoting substances. Substances which promote growth include, for example, growth promoters, trace elements, such as iron, zinc, manganese and the like, or individual amino acids.

If the promoter of the structural gene is regulated, the composition of the growth medium has to be adapted in order to obtain maximum levels of mRNA transcripts, e.g. when using the PH05 promoter in S. cerevisiae, the growth medium must contain low concentration of inorganic phosphate for derepression of this promoter.

The cultivation is carried out by employing conventional techniques. The culturing conditions, such as temperature, pH of the medium and fermentation time are selected in such a way that maximal levels of the desired polypeptide are produced. In general growth is performed under aerobic conditions in submerged culture with shaking or stirring at a temperature of about 25° to 35°C, at a pH value of from 4 to 8, for example at approximately pH 7, and for about 4 to 20 hours, preferably until maximum yields of the desired proteins are reached.

In the case of transformed plant cells the plant protoplasts are cultured in vitro or, alternatively, calli or whole plants are generated.

It is possible that the foreign polypeptide performs its function within the transformed cell and under these circumstances isolation is unnecessary.

The isolation and purification of the expressed polypeptide, if desired, is performed according to methods known in the art. After the transformed cells have been grown to a satisfactory cell density, the first step for the recovery of the expressed protein consists in liberating the protein from the cell interior. In most procedures the cell wall is first removed by enzymatic digestion, e.g. with glucosidases. Subsequently, the resulting spheroplasts are treated with detergents, such as Triton. Alternatively, mechanical forces, such as shearing forces (for example X-press, French-press) or shaking with glass beads, are suitable for breaking cells. The resulting protein mixture is enriched for the desired polypeptide by conventional means, such as removal of most of the non-proteinaceous material by treatment with polyethyleneimine, precipitation of the proteins by saturating the solution with ammonium sulphate or trichloroacetic acid, gel electrophoresis, dialysis, chromatography, for example, ion exchange chromatography, size-exclusion chromatography, HPLC or reverse phase HPLC, molecular sizing on a suitable Sephadex^® column, or the like. The final purification of the pre-purified product is achieved, for example, by means of antibody affinity chromatography.

A mixture of glycosylated and unglycosylated proteins may be separated, for example, by chromatography on a concanavalin-A Sepharose^® column. Unglycosylated products will pass through the column whereas glycosylated products will selectively adsorb and can be eluted by conventional means, e.g. α-methylmannoside in combination with a chaotropic agent, such as KSCN.

It is also possible to remove glycosyl residues enzymatically, e.g. by the action of endoglycosidase H. This method permits the production of unglycosylated products in substantially pure form.

The invention concerns furthermore the polypeptides whenever prepared according to the methods of the present invention.

The invention concerns also the polypeptides obtainable according to the inventive process. The invention concerns especially the eukaryotic hybrid vectors, the transformed eukaryotic cells and the processes for their preparation as well as the method for producing the polypeptides as described in the Examples.

The following Examples serve to illustrate the present invention but should not be construed as a limitation thereof.

Experimental part

The following abbreviations are used in the Examples:

BSA: bovine serum albumin

DTT: 1,4-dithiothreitol (1,4-diraercapto-2,3-butanediol)

EDTA: ethylenediaminetetraacetic acid

SDS: sodium dodecyl sulphate

TNE: solution containing 100 mM NaCl, 10 mM Tris·HCl

(pH 7.5), and 1 mM EDTA.

Tris·HCl tris-(hydroxymethyl)-aminomethane, pH adjusted with HCl

TE: solution containing 10 mM Tris·HCl (pH 7.5) and 1 mM EDTA

Example 1 : Transformation of Saccharomyces cerevisiae with linear extrachromosomal rDNA of Physarum polycephalum

a) Preparation of the linear extrachromosomal rDNA from Physarum polycephalum (P.p)

P.p. M₃CV III (Dr. W. Dove, McArdle Institute for Cancer Research, Univerity of Winsconsin, Madison) is grown in a liquid culture medium as described by Daniel and Baldwin (1964)(4.) The rDNA is isolated as described by Behrens et al. (1982)(5.). b) Preparation of spheroplasts from Saccharomyces cerevisiae (S.cer.)

Spheroplasts of S.cer. strains RH 218 are prepared as described (Hinnen et al., 1978, 30).

c) Tranformation

100 μl of spheroplasts (2.10 cells/ml) in 10 mM Tris/HCl pH 7.5, 10 mM CaCl₂, 1M sorbitol, are mixed with 10μl of the rDNA (lμg/μl ) obtained according to a) and incubated for 20 min at room temperature. 1 ml of 20 % polyethylene glycol 4000 (Sigma) is added and the mixture is kept for 30 min at room temperature. Then the cells are centrifuged at 1000 x g for 5 min and resuspended in 0.5 ml of 1 M sorbitol. The cells are mixed with 10 ml YPD regeneration agar, kept at 50°C, (YPD regeneration agar: 20 g/l Bacto peptane, 10 g/l Bacto yeast extract, 20 g/l glucose, 182 g/l sorbitol, 30 g/l agar) and poured onto YPD agar plates (YPD agar: 20 g/l Bacto Peptone, 10 g/l Bacto Yeast Extract, 20 g/l glucose, 20 g/l agar). The plates are incubated at 30°C for 3-4 days.

Example 2: Test for the presence of linear extrachromosomal rDNA in the S.cer. cells.

Cells from the lawn of the transformation plates (Example lc) are spread onto new YPD plates at a cell density which allows growth of individual colonies and incubation takes place for 2 days at 30°C. Cell material from a single colony is picked to inoculate a 100 ml YPD liquid culture (as YPD agar plates, but without agar, see 1c). Cells are grown overnight at 30°C at 200 rpm. For the isolation of total DNA the cells are centrifuged at 5000 x g for 5 min and resuspended in 10ml 1M sorbitol, 50 mM EDTA, 14mM DTT (dithiothreitol). Then 1 mg of Zymolyase 60'000 (Miles) is added and incubation takes place at 30°C for about 60 min. The spheroplasts formed are centrifuged and resuspended into 10 ml of 1 M sorbitol, 50 mM EDTA. Proteinase K (Boehringer, Mannheim) is added at a concentration of 0.2 rag/ml and the mixture incubated for 30 min at 37°C. The spheroplasts are centrifuged as before and resuspended in 1 % SDS, 10 mM Tris/HCl pH8, 10 mM EDTA, 0.1 mg/ml Proteinase K, and incubated 1 hour at 37°C. The solution is extracted twice with equal volumes of phenol and once with chloroform. The DNA is precipitated with 2 volumes of ethanol and resuspended in 3 ml 10 mM Tris/HCl, pH 7.5, 1 mM EDTA. 3.9 g of CsCl is added to obtein a refractive index of the solution of n = 1.401. The solution is centrifuged in a TV865 Sorval vertical rotor for 18 hours at 38000 rpm at 15°C. The CsCl gradient is fractionated by puncturing the tube from the bottom (20 fractions/tube). 20 μl of each fraction is transferred to nitrocellulose filter paper (Schleicher and Schϋll) and the filters are immersed into 1.5 M NaCl, 0.1 M NaOH, washed in 2xSSC (see below) and incubated at 80°C the filters are incubated at 80°C under vacuum (dot analysis). Filters containing DNA from transformed and untransformed cells are hybridized by 32P-labelled plasmid DNA prepared by nicktranslation (Maniatis et al. (7), p.109) (hybridization conditions see below). The plasmid DNA used is a 2.0 kbp Sal I fragment from the nontranscribed internal spacer region. The analysis shows that the rDNA isolated from S.cer. sediments in a CsCl gradient at a density which corresponds to the density of authentic, linear extrachromosomal rDNA obtained from P.p..

Fractions from the CsCl gradient are diluted 10-fold with 10 mM Tris/HCl pH8, 1 mM EDTA (=TE) and the DNA is precipitated by adding two volumes of ethanol.

The DNA is resuspended in 100 μl TE. 2μg DNA is digested with Taq I in a volume of 40 μl according to the specifications given by the supplier (Boehringer). The DNA is separated by agarose gel electrophoresis (1 % agarose gel) in TBE buffer (Maniatis et al. (7), p. 156) transferred to nitrocellulose filter paper according to Southern (Manitatis et al. (7), p. 382) and hybridized with the same radioactively labelled plasmid DNA fragment as described above. The hybridization buffer consists of: 0.016 % bovine serum albumin, 0.016 % Ficoll, 0.016 polyvinylpyrrolidone, 0.6 M NaCl, 0.1 Tris/HCl pH 8.0, 0.004M EDTA, 0.08 % NaH₂PO₄, 40 % formamide, 0.16 % SDS, 0.1 mg/ml calf thymus DNA. The hybridization is performed for 48 hours at 37°C. The filters are incubated in 0.1xSSC at 65°C for 2 hours (1xSSC is 0.15 M NaCl 0.015 M Na citrate pH 7.2). The results show that the restriction fragments correspond to the fragments of authentic rDNA of P.p.. The same experiment can be performed with S. cer. JH-D5 (J. Carbon, University of Santa Barbara)

Example 3: Tranformation of Schizosaccharomyces pombe with linear extrachromosomal rDNA of Physarum polycephalum

S. pombe strain h leu 1-32 (obtained from U. Leupold, Institut fur Mikrobiologie, Universitat Bern) are grown in 100 ml YEL medium (5 g/l Bacto Yeast Extract, 30 g/l glucose) and spheroplasts are prepared as described by Beach and Nurse (6). The transformation protocoll is as described for S.cer., except for the spheroplast buffer (1.2 M sorbitol, 50 mM Na citrate pH 5.6, 10 mM CaCl₂, 10μg/ml calf thymus DNA) . After the PEG incubation step the spheroplast/DNA mixture is suspended in 10 ml of YEL medium containing 2 % agar (kept at 50°C) and pured onto YEL plates (5 g/l Bacto Yeast Extract, 30 g/l glucose, 20 g/l agar) Incubation is at 30°C for 4 days.

Example 4: Test for the presence of linear extrachromosomal rDNA in S.pombe cells

DNA derived from purified single colonies from the transformation lawn is isolated as described for S.cer. (see 2) except for the use of a Trichoderma harzianum extract, SP234 (Novo Enzymes), as a source of glucanases (final concentration is 5 mg/ml of SP234) in the citrate buffer system described under Example 3.). For the demonstration of the presence of rDNA of P.p. in transformed cells of S. pombe the dot analysis described under Example 2. is used. The same experiment can be performed with S. pombe h-ura4-294 (U. Leupold, Universitat Bern). Example 5: Transformation of Cephalosporium acremσnium with linear extrachromosomal rDNA of Physarum polycephalum

C. acremonium strain ATCC 11550 (American Type Culture Collection) is grown (25°C, 250 rpm) in 100 ml filtrated 48c medium (11.8 g/l Cornsteep, 20 g/l sucrose, 4.5 g/l ammonium acetate, 0,5 g/l CaSO₄, 0.5 g/l MgSOO. About 1 g of mycelium is obtained and washed 5 times with 50 ml 0.7 M KCl, 0.05 M KH₂PO₄, pH 6.0 (centrifugation for 5 min at 2000 x g). The mycelium is resuspended in 50 ml Ll/KClKH₂PO₄ pH 6.0 [=^*- 1 g LI enzyme/50 ml (BDH Chemicals), 0.7 M KCl, 0.05 M KH-PO^, pH 6.0] and incubated at 30°C for 4 hours (gives about 60-70 % spheroplasts as determined by microscopic analysis). The spheroplasts are removed from undigested mycelium by filtration through glass wool, and washed four times by centrifugation and resuspension in 50 ml 0.7 M KCl, 0,05 M KH^PO,, pH 6.0. Finally a total of 4 . 10⁸ spheroplasts are resuspended in 1.2 ml CST (10 mM CaCl₂, 2 M sorbitol, 25 mM Tris/HCl pH 7.8). Aliquots of 300 μl (10⁸ cells) are mixed with 7μl of TE containing 10 μg of rDNA of P.p. (control samples are applied without rDNA). The solutions are incubated at room temperature for 20 min, then 5 ml of 50 % PEG 4000 (in H₂O) is added and incubation takes place for 20 min at 30°C. Finally, 40 ml 0.7 M KCl, 0,05 M KH-P0₄, pH 6.0 is added and the cells are centrifuged at 2000 x g for 10 min, resuspended in 20 ml of the same buffer, centrifuged again and resuspended again in 0.5 ml of the same buffer.

Aliquots are mixed with 10 ml regeneration agar [30 g/1 Mycophil (Becton, Dickinson and Company), 4 g/l Bacto yeast extract, 90 g/l KCl, 25 g/l agar], kept at 50°C, and poured onto agar plates of the same composition. The plates are incubated at 25°C for 9 days. Example 6: Test for the presence of linear extrachromosol rDNA in C.acremonium

A piece of mycelium (3cm x 3cm) is cut from the transformation plates and incubated in 5 ml of 48 c medium (see Example 5) at 25°C and 250 rpm. After 3 days the culture is used to inoculate 100 ml of the same medium and incubation takes place for another 3 days. Then, 5 ml of this culture is used to inoculate 100 ml of filtrated 48 c medium and incubation is for 24 hours under the above conditions. Spheroplasts are prepared as described under Example 5, except that they are finally resuspended in 2.5 ml lysis buffer (50 mM Tris/HCl pH7.5, 5 mM EDTA, 1 % SDS, 20 mg/ml proteinase K). 10 μl diethylpyrocarbonate is added and the mixture is incubated for 4 hours at 37°C with 50 rpm. The solution is heated for 20 min at 65°C and then centrifuged at 25'000 x g at room temperature. The supernatant is precipitated with two volumes of ethanol and the sediment is resuspended in 3ml TE. Fractionation on a CsCl density gradient and the detection of the rDNA is as described for the rDNA of S.cerevisiae (see Example 2.).

Example 7: The use of P.p. linear extrachromosomal rDNA as a vector to introduce the tissue specific plasminogen activator (TPA) gene into S.cer..

Construction of rDNA/TPA hybrid

Five μg of plasmid p-JDB207/PH05 - TPA(IA) is digested with restriction endonuclease Hindlll (New England Biolabs) in a total volume of 50 μl, according to the instructions of the supplier. The DNA is extracted once by adding 50 μl of phenol. After three chloroform extractions the DNA is precipitated with ethanol and resuspended in 100 μl nick translation buffer (see Maniatis et al.(7), p. 112). After the addition of the four nucleotides dATP, dGTP, dCTP and TIP at a final concentration of 80 μM, 5 units of Klenow fragment of DNA polymerase I is added and the mixture is incubated as described (Maniatis et al. (7) p. 113). The mixture is extractd with phenol and chloroform as described above and the DNA is precipitated with ethanol. 1 μg of Bam HI linkers (5'-CGGATCCG-3' , New England Biolabs) are kinased in 50 μl of 6 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 4mM DTT, 0,5 M ATP and 35 U of T4 polynucleotide kinase (Boehringer) for 30 min at 37°C (see Maniatis et al. (7), p. 125) 0.9 μg of kinased linkers and 0.6 μg of blunt ended vector DNA are ligated overnight at room temperature in 25 μg of 60 mM TrisΗCl pH 7.5, 10 mM MgCl₂, 5 mM DTT, 3.5 mM ATP and 450 U of T4 DNA ligase (New England Biolabs). The ligated DNA is separated from excess linkers by isopropanol precipitation in the presence of 10 mM EDTA, 0.3 M sodium acetate pH 6.0 and 0.54 volumes of isopropanol. After 30 min of incubation at room temperature the DNA is sedimented by centrifugation. The pellet is air dried, resuspended in 50 μl Bam HI buffer (New England Biolabs.) and digested with Bam HI. The mixture is separated by electrophoresis in a 0.6 % soft agarose gel (Sigma) in TBE buffer. The 2.7 Kb DNA fragment is cut out from the agarose gel, extracted three times with phenol, two times with chloroform and then precipitated with ethanol. Five hundred μg of plasmid pBR322 is digested with Bam HI and 200 μg of the digested DNA is mixed with the above 2.7 kb Bam HI fragment. The DNA is ligated in 20 μl of 60 mM Tris-HCl pH 7.5, 10 mM MgCl₂, ImM ATP, 5 mM DTT and 400 U T4 DNA ligase at 15°C for 6 hours. Aliquots of 2 μl are added to 100 μl of calcium-treated, transformation competent E. coli HBlOl cells (Maniatis et al.(7), p. 250) and transformants are selected on LB agar plates containing 100 μg/ml ampicillin (LB: 10 g/1 Bacto Tryptone, 5 g/l Bacto Yeast Extract, 5 g/1 NaCl, 15 g/1 Agar). Ten colonies which are sensitive to 10 μg/ml tetracyclin are picked and plasmid DNA is isolated by the method of Holmes and Quigley, described by Maniatis et al. ((7), p. 366).

All isolates are found to contain a 2.7 kb Bam H fragment inserted within the tetracyclin resistance gene of pBR322. One isolate is picked and plasmid DNA is prepared as described (Maniatis et al.(7), p. 92). Twenty microgramm of plasmid DNA is digested in a total volume of 200 μl with restriction endonuclease Bam HI and the DNA is applied on a 0.6 % soft agarose gel (TBE buffer). The 2.7 kb fragment is cut out and phenolized two times. After two extractions with chloroform the DNA is precipitated with ethanol and resuspended in 20 μl of TE buffer.

One μg of the 2.7 kb Bam HI fragment is mixed with 10 μg of rDNA of P.p. which has previously been cut with restriction endonuclease Bgl II (supplier and digestion conditions: New England Biolabs.). The DNA is ligated in 50 μl of 60 mM Tris HC1 pH 7.5, 10 mM MgCl₂ ImM ATP, 5 mM DTT and 400 U T4 DNA ligase at 15°C over night. The reaction mixture is made 0.25 M in NaCl and the DNA is precipitated with ethanol. The DNA is resuspended in Bgl II buffer (New England Biolabs) and digested with Bgl II restriction enzyme for two hours. Then the DNA is again precipitated with ethanol and resuspended in the above ligation buffer. This ligation/digestion cycle is repeated four times until this DNA is used for a transformation experiment as described under Example 2.

Cells from the transformation plates are used to inoculate 20 ml of low P. medium (Meyhack et al. (8), 1982).

Cells from the low P. culture medium at a cell density of 1-2x10⁷/ml are collected by centrifugation in a Sorvall SS34 rotor for 10 min at 3000 rpm. The cells are washed in a buffer containing the salt components of the culture medium (i.e. without aminoacids, glucose, vitamines, traceelements). The cells are centrifuged at room temperature for 5 min at 3000 rpm. The sedimented cells are resuspended in a total volume of 4 ml of cold 66 mM sodium phosphate buffer pH 7.4 and 0.1 % (v/v) Triton X-100. The cell suspension is transferred to a 30ml Corex tube, 8 g of glass beads (0.4 mm in diameter) are added and the suspension is shaken on a Vortex Mixer (Scientific Instruments Inc., USA) at full speed for 4 min. and then cooled in an ice bath. More than 90 % of the cells are broken by this procedure. Cell debris and glass beads are sedimented by centrifugation for 10 min at 8000 rpm at 4°C in a Sorvall HB-4 rotor. The supernatant is transferred to Eppendorf tubes, frozen in liquid nitrogen and stored at -60°C. TPA activity is determined according to the method of Rånby (9) with slight modifications. D-Val-Leu-Lys-pNA (Kabi S-2251) is used as substrate. The absorption at 405 nm is corrected for unspecific cleavage and related to an urokinase standard.

The activity recovered corresponds to approximately 0.1 mg/l yeast cell culture.

The plasmid pJDB207/PHO5-TPA(IA) is produced as follows:

Example A: Construction of plasmid p30 (see fig. 4)

a) Elimination of the Ball restriction site in plasmid pBR322 3 μg of pBR322 are digested to completion with restriction endonucleases Ball (BRL) and PvuII (Biolabs) according to the recommbinations of the suppliers. The Ball/PvuII double digest of pBR322 results in two restriction fragments of 3738 dp and 622 bp in size. The two fragments are separated on a 1 % low melting agarose gel (Sigma) in TBE (90 mM TrisΗCl pH 8.3, 2.5 mm EDTA, 90 mM boric acid) buffer. The DNA bands are stained with ethidiumbromide and visualized under long wave UV light at 366 nm. The piece of agarose containing the 3738 bp fragment is cut out from the gel, liquified at 65°C, adjusted to 500 mM NaCl and incubated at 65°C for 20 min. One volume of phenol (equilibrated with 10 mM Tris«HCl pH 7.5, 1 mM EDTA, 500 mM NaCl) is added. The aqueous phase is reextrated twice with phenol and once with chloroform. The dNA is precipitated with 2.5 volumes of cold absolute ethanol and collected by centrifugation. The DNA pellet is washed with cold 80% ethanol and then dried in vacuum. The DNA is resuspended in TE at a concentration of 0.15 mg/ml.

The isolated 3738 bp DNA fragment has two blunt ends resulting from the Ball and PvuII double digests. The DNA is circularized by blunt end ligation. 0.6 μg of DNA are incubated over night at room temperature in 30 μl of 60 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 10 mM DTT, 4 mM ATP, and 900 U of T4 DNA ligase (Biolabs). 5 μl of clacium treated, transformation competend E. coli HB101 cells, prepared by the method of Mandel et al. (10). The mixture is kept on ice for 5 min, then incubated for 2 min at 37°C and left 10 min at room temperature before plating on LB agar plates containing 100 μg/ml of ampicillin. Six amp colonies are picked an grown individually in 100 ml of LB (as above but without agar) medium containing 100 μg/ml ampicillin. Plasmid DNA is prepared from the cells using the procedure described by Maniatis et al. (p. 92 (7)). Restriction digests with Haelll (purchase from Biolabs, digestion conditions as suggested by supplier), PvuII and Ball of the plasmids are analyzed on a 1.5% agarose gel in TBE buffer. The restriction pattern and the predicted size of the newly formed junction fragment indicates that the plasmids are identical and contain all the pBR322 sequences except for the Ball - PvuII fragment. These plasmids lack the Ball restriction site and are referred to as pBR322 Δ Ball.

b) Cloning of a yeast 5.1 kb BamHI restriction fragment containing PH05 and PH03 into pBR322 Δ Ball

pJDB207/PH05,PH03 (Meyhack et al. (8) contains a yeast 5.1 BamHI insert with the genes for regulated and constitutive yeast acid phosphatase (PH05 and PH03) . pJDB207/PH05,PH03 as well as pBR322 Δ Ball are digested with restriction endonuclease BamHI. After complete digestion the enzyme is inactivated for 2 min at 65°C. Both DNAs are precipitated by ethanol and resuspended in 10 mM Tris'HCl pH 3.0 at a concentration of 0.2 mg/ml each. 0.5 μg of each of the two BamHI-digested DNAs are combined and ligated in 20 μl of ligation buffer (as suggested by New England Biolabs), containing 300 U of T4 DNA ligase, for 20 hrs at 15°C. 5 μl aliquots of the ligation mixture are added to 50 μl of calcium-treated E. coli HBlOl cells and transformation is carried out as described in Example Aa.

The transformed E. coli cells are tested for their resistance towards ampicillin and tetracyclin. Eight amp^R, tet^S colonies are isolated and grown in 100 ml of LB medium containing 100 μg/ml of ampicillin. Plasmid DNA is isolated from the cells (Maniatis et al., p. 92 (7)). Restriction digests with BamHI show that 4 plasmids contain a 5.1 kb insert besided the 3.7 kb vector fragment (pBR322 Δ Ball). Restriction digests with Sall (New England Biolabs) determine the orientation of the inserted 5.1 kb fragment: two plasmids have the insert oriented as shown in figure 4. One of them is referred to as p30. The direction of transcription of the PHO5, PHO3 genes in the 5.1 kb insert is anticlockwise as indicated in figure 4.

Example B: Construction of an expression plasmid containing the PHO5 promoter and PHO5 transcription termination signals (see fig. 5)

a) Elimination of the EcoRI restriction site in plasmid p30:

Five μg of p30 DNA (cf. Example A) are digested to completion with restriction endonuclease EcoRI (Boehringer). In order to fill in the resulting sticky ends, 1 μg of EcoRI digested p30 in 50 μl of 50 mM NaCl, 10 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT, 0.25 mM dATP and 0.25 mM dTTP is incubated for 30 min 37°C with 1 unit of DNA polymerase (Klenow large fragment, BRL) . The DNA recovered fro methanol precipitation is ligated as usual and used for transformation of competent E. coli HBlOl cells as described in Example A. Clones that are resistant to EcoRI digest are referred to as p30(EcoRI^R).

b) Isolation of a 0.37 kb Sau3A-PstI PHO5 transcription termination fragment.

The PHO5 transcript has been mapped by SI nculease mapping (25). The signals for transcription termination have been shown to bel located in a 0.37 kb Sau3A-PstI fragment of the PHO5 gene. The nucleotide sequence of the Sau3A-PstI fragment is given in fig. 6. Five μg of pJDB207/PHO5,PHO3 DNA (8) are digested to completion with restriction endonucleases Sau3A and Pstl. The restriction fragments are separated on a vertical 1.5% low melting agarose gel in TBE buffer. The 0.37 kb Sau3A-PstI fragment is localized by ethidiumbromide staining and a gel block as small as possible is cut out containing this DNA fragment.

c) Cloning of the Sau3A-PstI PHO5 fragment in M13mρ9:

M13mp9 phage DNA is a useful cloning vector with a cluster of unique restriction sites (11). Five μg of M13mp9 DNA are digested to completion with restriction endonucleases BamHI and Pstl. The larger 7.2 kb DNA fragment is separated from a very small fragment (8 dp) on a 0.5% low melting agarose gel. The gel block containing the large DNA fragment is cut out of the gel. Gel blocks with the 0.37 kb Sau3A-PstI fragment of pJDB207/PHO5,PHO3 (cf. Example Bb) and the 7.2 kb BamHI-PstI fragment of M13mp9 are liquified at 65°C, mixed in about equimolar amounts and diluted with H₂O to lower the agarose concentration to 0.3%. Ligation is carried out in a 200 μl solution containing 60 mM Tris.HCl pH 7.5, 10 mM MgCl₂, 10 mM DDT, 1 mM ATP and 600 units of T4 DNA ligase (Biolabs). Transduction of competent cells of the strain E. coli JM101 (Ca⁺⁺) is done according to the manual "M13 cloning and DNA sequencing system" published by New England Biolabs. Phages from a number of white plaques are grown and analyzed for the size of their DNA insert by cleavage with restriction endonucleases EcoRI and Pstl.

A M13mp9 derived clone containing the Sau3A-PstI PHO5 transcription termination fragment is isolated and referred to as M13mp9/PHO5(Sau3A-PstI). d) Cloning of the PHO5 transcription termination fragment in p30(EcoRI^R)

The original PHO5 transcription termination fragment cloned in phage

Ml3rap9 (M13mp9/PHO5(Sau3A-PstI) ) is recloned as a Haelll-Hindlll fragment in plasmid p30(EcoRI^R) cleaved with Ball and Hindlll:

Ml3mp9/PHO5(Sau3A-PstI) DNA is cleaved to completion with restriction endonucleases Haelll and Hindlll. The resulting two DNA fragments are separated on a 1.5% vertical low melting agarose gel in TBE buffer. The 0.39 kb fragment is isolated in a gel block cut out of the gel. p30(EcoRI^R) DNA is digested with Ball and Hindlll. The large 3.98 kb fragment is separated on a 0.5% low melting agarose gel in TBE buffer and isolated by cutting a gel block containing the DNA fragment.

Gel blocks with the 0.39 kb Haelll-Hindlll transcription termination fragment and the 3.98 kb Ball-Hindlll fragment of p30(EcoRI^R) are melted at 65°C and mixed in about equimolar amounts. Ligation and transformation of competent E. coli HB101 cells are as described in Example A. DNA of transformed cells is analysed by cleavage with Ball and Haelll. A clone containing the PHO5 transcription termination fragment is further analyzed and referred to as p31 (see Figure 5).

Expression plasmid p31 contains the PHO5 promoter region with part of the signal sequence of PHO5 and adjacent to it a DNA fragment with the PHO5 transcription termination signals. Foreign coding sequences to be expressed in this vector may conveniently be inserted between promoter and transcription termination sequences.

Example C: Deletion of the PH05 signal sequence in the expression plasmid P31 (see figure 7) Expression plasmid p31 contains the PHO5 promoter sequence including the mRNA start sites, the translation start codon ATG of acid phosphatase and additional 40 nucleotides coding for part of the acid phosphatase signal sequence. In this construction, the nucleotides for the signal sequence and the ATG are eliminated by Ball31 digestion. EcoRI linkers are introduced to allow joining of the PHO5 promoter to mature TPA coding sequence.

a) Bal31 digestion of Ball cleaved plasmid p30 20 μg of p30 DNA (see Example Ab) are digested with restriction endonuclease Ball, resulting in 2 fragments of 3.7 and 5.1 kb. After extraction with with phenol/chloroform, the DNA are digested with 2 U of exonuclease Bal31 (BRL) in 100 μl of 20 mM Tris pH 8.0, 100 mM NaCl, 12 mM MgCl₂, 12 mM CaCl₂ and l mM EDTA. Aliquots of 2 μg DNA each are withdrawn after 15 sec, 30 sec, 45 sec. and 60 sec. of incubation at 30°C and are immediately mixed with 50 μl phenol and 60 μl TNE. After extraction with phenol/chloroform and ethanol precipitation, the DNA is resuspended in 10 mM Tris pH 8.0 at a concentration of 100 μg/ml. To analyse the extent of exonucleolytic cleavage by Ball30.5 μg of DNA from each time point are digested with endonuclease BamHI and analysed on a 1.5% agarose gel in Tris-borate buffer pH 8.3. On the average 70 pb are removed from each end of the fragment after 45 sec of Bal31 digestion. For further experiments DNA from the 45 second time point is used.

The annealed, double stranded EcoRI linkers are ligated with their blunt ends to the Bal31 treated DNA fragments. Half a microgram of Bal31 treated DNA (see Example Ca) is incubated for 16 hours at room temperature with a 50fold excess of kinased EcoRI linkers in 20 μl of 60 mM Tris pH 7.5, 10 mM MgCl₂, 10 mM DTT, 4 mM ATP and 600 U of T4 DNA ligase (Biolabs). After inactivation of the T4 DNA ligase (10 min at 65°C) the excess of EcoRI linkers is cleaved by 50 U of EcoRI (Boehringer) in a volume of 50 μl. The DNA is extracted with phenol/chloroform, precipitated by ethanol and resuspended in 10 mM Tris, 1 mM EDTA.

Restriction endonuclease EcoRI not only cleaves the terminally added EcoRI linkers of both Ball fragments (3.7 kb and 5.1 kb) but also at an internal EcoRI site in the 5.1 kb fragment giving rise to a 3.9 kb and a 1.2 kb fragment. The 3.7 kb and 3.9 kb fragments are separated from the 1.2 kb fragment on a 0.8 % low melting agarose gel (Sigma) in 90 mM Tris·HCl pH 8.3, 90 mM boric acid and 2.5 mM EDTA. The DNA bands are stained with ethidium bromide and visualized under long wave UV light at 366 nm. The two large DNA fragments of 3.7 kb and 3.9 kb are not separated. They are cut out of the gel in a single gel block and are extracted as described in Example 4a.

The linear fragments terminating in EcoRI sticky ends are circularized by ligation. About 0.25 μg of fragments are ligated in 100 μl of 60 mM Tris pH 7.5, 10 mM MgCl₂, 10 mM DTT, 1 mM ATP ad 600 U T4 DNA ligase for 4 hours at 15°C.

Ten μl aliquots of the ligation mixture are added to 100 μl of calcium treated, transformed competent E. coli HBlOl cells (see Example Aa). 35 transformed, amp colonies are grown individually in LB medium containing 100 μg/ml of ampicillin. Plasmid DNA is prepared according to the method of Holmes and Quigley (see 7, p. 366) and is analysed by EcoRI/BamHI double digestion.

c) Nucleotide sequence analysis to determine the position of the

EcoRI linker addition Most of the 35 clones will differ from each other in the position of EcoRI linker addition in the PHO5 promoter region depending on the degree of Bal31 digestion of the individual DNA molecules. For the nucleotide sequence analysis plasmid DNA is digested with EcoRI. After extraction with phenol/chloroform the restricted DNA is precipitated with ethanol. The DNA is dephosphiorylated and 5'-terminally labeled. The labeled DNA fragments are cleaved with a second restriction endonuclease, BamHI. The products are separated on a 0.8% low melting agarose gel. The 0.5-0.6 kb 5'-labeled EcoRI-BamHI fragment is isolated from low melting agarose as described in Example Aa. For the determination of the nucleotide sequence adjacent to the EcoRI linker the different DNA fragments are chemically degraded and the products are separated by polyacrylamide gel eletrophoresis as described by Maxam and Gilbert (12). The different clones and the position of the corresponding last nucleotide of the PHO5 sequence (then followed by an EcoRI linker) is listed in Tab. 1 (see also figure 8).

d) Isolation of a 0.53 kb BamHI-EcoRI fragemnt containing the PHO5R promoter:

Plasmid pR contains the PH05R promoter on a 534 bp BamHI-EcoRI fragment. According to the numbering in fig. 3a, the fragment covers PHO5 promoter sequences from nucleotide - 541 (BamHI site) to nucleotide - 10. An EcoRI linker, ligated to nucleotide - 10 (see Example Cb) contributed two G-residues upon EcoRI cleavage.

Plasmid pR is digested with restriction endonucleases BamHI and EcoRI. The 0.53 kb BamHI-EcoRI fragment is separated on a 0.5% low melting agarose gel and isolated as described in Example Aa. The nucleotide sequence is given in fig. 9.

In an analogous manner plasmid pY is digested and a 0.53 kb BamHI-EcoRI fragment is isolated containing the PHO5Y promoter. The nucleotide sequence is given in fig. 10.

e) Replacement of the Sall-EcoRI fragment in plasmid p31 by a Sall-EcoRI fragment of the new constructions

Five μg of plamid P31 (cf. Example Bd) are digested with restriction endonuclease Sail. The restricted DNA is precipitated with ethanol and resuspended in 50 μl of 100 mM Tris pH 7.5, 50 mM NaCl, 5 mM MgCl. The DNA is digested with EcoRI to completion. The restriction fragments are separated on a 0.8% low melting agarose gel in Tris-borate-EDTA buffer pH 8.3. A 3.5 kb DNA fragment is isolated in a small gel block containing the DNA band.

Five μg each of clones pR and pY (cf. Table 1 and fig. 8) are digested with Sall and EcoRI in the same way as described above. The 0.8 kb DNA fragments are isolated in small blocks of low melting agarose gel.

0.67 μg of the 3.5 kb Sall-EcoRI fragment of vector p31 is ligated to 0.34 μg of the 0.8 kb Sall-EcoRI fragment of plasmid pR or pY, raespectively. Appropriate gel blocks, containing the DNA fragments are mixed and melted at 65°C. The liquified gel is diluted three times. Ligation is performed in a total volume of 240 μl of 60 mM Tris pH 7.5, 10 mM MgCl₂, 10 mM DTT, l mM ATP with 750 U of T4 DNA ligase (Biolabs) overnight at 15°C. A 2 μl aliquot of each of the ligations is added ot 100 μl of calcium treated, transformation competent E. coli HB101 cells (see Example Aa).

8 transformed, amp^R colonies each are grown individually in LB medium containing 100 μg/ml ampicillin. Plasmid DNA is analysed by restriction analysis. The clones of each group are identical. One clone each is further used and referred to as p31R or p31Y, respectively (fig. 7).

Example D: Preparation of the TPA gene

a. Preparation of mRNA Falcon cell culture flasks (175 cm²) are seeded with a primary inoculum of 10 x 10⁶ HeLa cells and maintained in 25 ml of Dulbecco's modified Eagle's medium (DME) supplemented with 5% fetal bovine serum. The medium is changed every three days until confluency is attained.

Confluent monolayer cultures are washed twice with PBS (phosphatebuffered saline: 80 g NaCl, 2 g KCl, 14.4 g Na₂HPO₄ and 2 g KH₂PO₄ per 1 solution) and subsequently maintained for 16 hours in DME supplemented with 100 ng/ml TPA. Total RNA is extracted as described (13), applying lysis buffer directly to the cell monolayers. Poly(A)-rich sequences are isolated from total RNA according to the method of Nagamine et al. (14).

Sucrose gradient centrifugation is performed in a Beckman ultracentrifuge equipped with a SW-41 rotor. 100 μg of poly(A) RNA in 200 μl H₂O are centrifuged through a 15-30% sucrose gradient in 0.02 M Tris.HCl, pH 7.4, 0.1% SDS, 0.01 M EDTA, 0.04 M NaCl at 30,000 rpm for 12 hours at 18°C. The gradient is fractioned from the bottom into 36 fractions and RNA is precipitated by the addition of NaCl to 0.2 M and 2.5 volumes EtOH. After an overnight incubation at -20°C, RNA is recovered by centrifugation and dissolved in 20 μl of H₂O.

Stage six oocytes are selected by visual inspection, maintained in modified Barth's medium and injected with mRNA from the sucrose gradient fractions essentially as described (15). Plasminogen activator secretion by injected oocytes is demonstrated by radial caseinolysis as described by Nagamine et al. (14). Maximal TPA secrection is seen in oocytes injected with the 21-23S fractions.

b. cDNA synthesis and molecular cloning

HeLa poly(A) RNA enriched for TPA mRNA is copied into cDNA. Eight μg of 50 mM Tris.HCl, pH 8.3, 75 mM KCl, 8 mM MgCl₂, 1% v/v ethanol,

0.2 mM ETDA, 25 μg/ml oligo dT-_12-18; 0.5 mM each of dNTP and 12 μCi of [α³²P]dCTP (New England Nuclear). The solution is cooled to 0°C and 3 mM sodium pyrophosphate, 1 mM DTT and 144 units reverse transcriptase (Life Sciences Ltd) are added. After 60 min at 39°C, 32 additional units of reverse transcriptease are added. After 100 min incubation the reaction is stopped by the addition of SDS to 0.2%, EDTA to 15 mM. Incorporation of [α³²P]dCTP into cDNA is assessed by trichloroacetic acid precipitation of aliquots of the reaction mixture. 1.15 μg cDNA is synthesized from 8 μg RNA (14.4% copy). Following two extractions with chloroform-isoamylalcohol

(24:1 v/v), the aqueous phase is desalted on a 4 ml column

(5 x 0.5 cm) of Sephadex G50 fine by centrifugation in 20 mM

Tris-ΗCl, pH 9, 100 mM NaCl, l mM EDTA. Void volume fractions are pooled and NaOH is added to 0.3 N. After 12 hours at 20°C, HCl is added to 0.3 N and nucleic acids are precipitated by the addition of

3 volumes ethanol. Second strand synthesis is performed in a 200 μl solution containing 200 mM N-(2-hydroxyethyl)-piperazine-N'-ethane¬

2-sulfonic acid (Hepes), pH 6.9, 30 mM KCl, 10 mM DTT, 10 mM MgCl₂,

0.5 mM each of dATP, dCTP, dTTP and 25 units Klenow fragment per μg cDNA. After 2 hour incubation at 25°C the reaction is stopped by the addition of SDS to 0.1% and the solution desalted on a 4 ml column of Sephadec G50 fine by centrifugation in 30 mM sodium acetate, pH 4.5, 0.2 m NaCl, 3 mM ZnCl₂, 0.1% SDS. 2.5 units/ml nuclease Sl are added to the void volume fractions and the solution incubated at 37°C for 30 min. The solution is extracted with phenol-chloroformisoamylalcohol (24:24:1, v/v) and nucleic acid in the aqueous phase precipitated with 0.3 M NaCl and 3 vol. ethanol. 76% of the [α ³²P]dCTP incorporated into the first strand remain precipitable by trichloroacetic acid following Sl digestion, and analysis on an alkaline agarose gel (16) shows double-stranded cDNA ranging in size from 500 to over 3000 bp. The ds cDNA is size-fractionated on a 5-20% sucrose gradient in 10 mM TrisΗCl, pH 7.4, 100 mM NaCl, 1 mM EDTA, 0.1% SDS. After 10 hours centrifugation at 12°C in a SW41 rotor at 39K, those fractions containing the largest size cDNA (43% of total samples) are pooled and precipitated by the addition of NaCl to 0.3 M, 5 μg/ml BSA and 3 volumes ethanol. 25 ng of sizefractionated cDNA are tailed with dC as described (17) in a 85 μl reation containing 450 units/ml terminal transferase, 0.1 M potassium cacodylate, pH 7.5, 2 mM CoCl₂, 1 mM EDTA, 0.1 mM DTT and 5 μM [α³²P]dCTP (20 μCi). After 3 min at 30°C the reaction is stopped by the addition of EDTA to 10 mM, SDS to 0.2% w/v and tRNA to 100 μg/ml. The solution is extracted twice with chloroform-isoamylalcohol and desalted on a 5ml column of Sephadex G50 fine in 20 mM Tris.HCl, pH 9, 100 mM NaCl, 1 mM EDTA. Void volume fractions were pooled and nucleic acid precipitated by the addition of NaCl to 0.3 M, 25 μg/ml tRNA and 3 volumes EtOH. 20 ng tailed, ds cDNA is annealed to 60 ng pBR322 tailed with dG at the Pstl site (18) in 20 μl 0.4 M NaCl, 10 mM TrisΗCl, pH 7.5, 1 mM EDTA for 10 min at 65°C, 2 hours at 45°C, 2 hours at 22°C and then slowly cooled to 4°C overnight. The annealed cDNA is mixed at 0°C with 100 μl suspension of competent E. coli HB101/LM1035 (19).

Following 15 min incubation at 0°C and 5 min at 37°C, 1.9 ml LB is added to the bacterial suspension and incubation continued at 37°C for 2 hours. Cells are plated onto LB agar plates containing 10 μg/ml tetracycline. 100 transformants/ng vector are obtained with a background frequency of re-annealed vector of 13%. 5400 transformants are toothpicked from isolated colonies into 96 well microtiter plates containing 200 μl LB and 10 μg/ml tetracycline, grown overnight at 37°C, then stored frozen at -80°C after the addition of glycerol to 50% v/v.

c Screening for TPA DNA sequences

Two synthetic oligonucleotides having the sequences

d5'-GGCAAAGATGGCAGCCTGCAAG-3' and d5'-GCTGTACTGTCTCAGGCCGCAG-3'

of human tissue plasminogen activator cDNA (20) are synthesized by the modified tri-ester method (21), purified by reverse-phase HPLC, and preparative polyacrylamide electrophoresis. The oligonucleotides are labelled with [γ³²P]ATP (New England Nuclear) on the 5' terminus using T4 kinase to a specific activity of 1 x 10⁶ cpm

(Cherenkov)/pmole (12).

Microtiter plates are thawed and transformant cultures are transferred in a 6 by 8 grid array to 82 mm Millipore HATF nitrocellulose filters, grown for 18 hours on LB agar plates containing 10 μg/ml tetracycline, and plasmids are amplified for 12 hours on LB agar plates containing 10 μg/ml tetracycline, 10 μg/ml chloroamphenicol. DNA is fixed to the filters by blotting each filter on Whatman 3M paper saturated with 10% SDS for 3 min; 0.5 N NaOH, 1.5 M NaCl for 5 min; 0.5 M Tris.HCl, pH 8, 1.5 M NaCl for 5 min; and 2xSSPE (SSPE: 0.15 M NaCl, 10 mM NaH₂PO₄, pH 7.4, 1 mm EDTA) for 5 min (7). Filters are air dried, then baked under vacuum at 80°C for 2 hours. Baked filteres are washed 2 hours at 37°C in 50 mM Tris.HCl, pH 7.5, 10 mM EDTA, 1 M NaCl, 1% SDS to remove adherent bacterial debris. Hybridization and washing is done as described by Nagamine et al. (14), using the empirical relationships between nucleotide length, G+C content and Tm that have been determined by Suggs et al. (22) and Smith (23). Filters are prehybridized (2 hours) and hybridized (12 hours) at 60°C in 1.6 ml filter of 900 mM NaCl, 60 mM NaH₂PO₄, pH 7.4, 7.2 mM EDTA (6xSSPE), 0.1% w/v each BSA, polyvinylpyrrolidone, Ficoll 400 (5 x Denhardt's solution), 200 μg/ml tRNA and 0.1% w/v SDS. A mixture of both ³²P-labelled oligonucleotides at o.3 pmol/ml are added to the pre-hybridization solution. Following hybridization, filters are washed in 6xSSC (SSC: 0.15 mM NaCl, 15 mM trisodium citrate) for 3x10 min at 4°C and 3x10 min at 60°C. Dried filters are exposed to Kodak XB5 X-ray film with a Dupont intensifying screen for 36 hours at -70°C. The microtiter wells that produce positive hybridization signals are identified and single colonies from these cultures are rescreened as above, hybridizing duplicate filters to each probe separately, and using 63°C for hybridization and washing the filters. Colonies showing positive signals with both sets of probes are picked from the master filter and grown overnight at 37°C in LB containing 10 μg/ml tetracycline. One of these colonies is referred to as pW349F. Plasmid preparations are obtained using the modified alkaline lysis method (24) followed by CsCl equilibrium centrifugation.

DNA of plasmid pW349F is analysed by digestion with restriction endonucleases Pstl and Bglll. The restriction fragments are of the expected sizes. It is therefore concluded that plasmid pW349F (7.1 kb) contains the complete structural gene for human tissue plasminogen activator.

Example E: Construction of plasmid pJDB207./PHO5-TPA( 1A)

The PHO5 promoter and the PHO5 signal sequence are joined in frame to the coding sequence for mature TPA. A PHO5 transcription termination signal is also included in the construction.

a) Construction of Ml3mp9/PHO5Bam-Sal (see fig. 11)

A 623 bp BamHI-Sall fragment containing the PHO5 promoter is obtained by cutting 2 μg of pBR322/PHO5Bam-Sal (see fig. 3) with the restriction endonucleases BamHI and SaIl (both from Biolabs) under the conditions indicated by the supplier. 2 μg of replicated form (RF) of the single stranded phage vector M13mp9 (11) is digested with the same enzymes. Both DNA preparations are loaded on a 0.6% soft agarose gel as described in Example B. A 623 bp fragment derived from plasmid pBR322/PHO5Bam-Sal and a 7.2 kb band derived from M13mp9 are extracted as described in Example Be 150 ng of the 623 bp fragment and 150 ng of the 7,2 kb Ml3mp9 DNA fragment are ligated with 200 units of T₄ DNA ligase (Biolabs) for 4 hours at 15°C in 60 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 10 mM DTT and 1 mM ATP.

E. coli strain JM 101 is transformed as described by Messing (11) and 12 white plaques are picked and analysed by restriction analysis of the RF plasmid (25). All of the analysed clones contain the 623 bp fragment inserted into the Barn-Sal site of Ml3mp9. A single isolate is selected and called Ml3mp9/PHO5Bam-Sal (see fig. 11).

b) Joining of the TPA protein coding sequence to the PHO5 signal sequence (see fig. 11)

2 μg of plasmid pW349F (cf. Example D) is digested with Bglll endonuclease (New England Biolabs) under conditions given by the supplier. The DNA is ethanol precipitated and resuspended in 6 mM Tris-HCl, pH 7.5, 50 mM NaCl, 6 mM MgCl₂. The 3' recessed termini of the DNA are filled using the Klenow fragment of E. coli DNA polymerase. The reaction takes place in a total volume of 50 μl with 2 units of enzyme in the presence of 80 μM of dATP, dGTP, dCTP and dTTP for 30 min at 37°C. The incubation is stopped by phenol extraction and the DNA is precipitated with ethanol.

2 μg of RF plasmid Ml3mp9/PHO5Bam-Sal is digested with restriction endonuclease Kpnl (Biolabs) as indicated by the supplier. The DNA is precipitated with ethanol and resuspended in 10 μl of 200 mM NaCl, 1 mM ZnSO₄ and 60 mM Na-acetate pH 4.6. One unit of S₁ exonuclease is added and the mixture is incubated for 60 min at 37°C. The reaction is stopped by phenol extraction, the DNA is ethanol precipitated and resuspended in 50 μl of 50 mM Tris.HCl, pH 7.5, 1 mM MgCl₂. 10 units of calf intestine alkaline phosphatase (Boehringer) are added and the mixture is incubated for 60 min at 37°C followed by 60 min at 65°C. The DNA is purified by DE52 (Whatman) ion exchange chromatography and then precipitated with ethanol. 1.5 μg of Bglll digested plasmid pW349F treated with Klenow DNA polymerase (see above) is ligated to 0.5 μg of Kpnl cut, S. digested and phosphatase treated Ml3mp9/PHO5Bam-Sal in a volume of 10 μl using 900 units T₄ DNA ligase in a buffer as described above, except that 4 mM ATP are used and the incubation takes place for 18 hours at room temperature. E. coli JM 101 cells are transformed, 6 white plaques are selected and the single stranded phage template is prepared as described (25). DNA sequencing of the junction between the 2.0 kb Bglll fragment and the M13mp9 vector is performed using the dideoxy chain termination method (26). The following oligodeoxynucleotide primer is used:

5' AGTATGGCTTCATCTCTC 3'

The oligodeoxynucleotide is synthesized by the phosphotriester method (27,28). It hybridizes within the PH05 promoter and allows elongation by the Klenow fragment of E. coli DNA polymerase across the desired DNA junction point. One correct isolate is obtained which has the following DNA sequence arrangement (starting from the ATG of the PHO5 protein coding sequence):

ATG TTT AAA TCT GTT GTT TAT TCA ATT TTA GCC GCT TCT TTG GCC AAT GCA GGA TCT TAC CAA GTG

PHO5 protein coding sequence TPA protein coding sequence

This construction is called Ml3mp9/PHO5-TPA (see fig. 11) c) Construction of a shortened PHO5 transcription termination fragment (see fig. 12)

10 μg of p31R plasmid DNA ( see fig. 7) are digested with restriction enzyme Smal, the DNA is extracted with phenol and ethanol precipitated. The DNA is resuspended in 10 mM Tris«HCl pH 8.0 at a concentration of 0.5 mg/ml. 10 μg of the Smal cleaved DNA are digested with 2 U of endonuclease Bal31 (BRL) in 100 μl of 20 mM Tris pH 8.0, 100mM NaCl, 12 mM MgCl₂, 12 mM CaCl₂ and 1 mM EDTA. Aliquots of 3 μg of DNA each are withdrawn after 90, 120 and 150 seconds of incubation at 30°C and are immediately mixed with 50 μl of phenol and 60 μl TNE. After extraction with phenol/ chloroform and ethanol precipitation, the DNA is resuspended in 10 mM Tris.HCl pH 8.0 at a concentration of 100 μg/ml. To analyze the exonucleolytic digestion of Bal31, aliquots of 0.7 μg of DNA from each time point are digested with Hindlll and analyzed by agarose gel electrophoresis. For further analysis the DNA from the 90 second time point is used. 2.2 μg of the plasmid DNA are incubated for 1 hour at 37°C with 2.8 U of Klenow DNA polymerase (large fragment of polymerasse I, BRL) in 35 μl of 60 mM Tris.HCl pH 7.5, 10 mM MgCl₂ and 0.1 mM dNTPs .

Three μg of Xhol linker (5'-CCTCGAGG-3' , Collaborative Research) are kinased in 50 μl of 6 mM TrisΗCl pH 7.5, 10 mM MgCl₂, 4 mM DTT, 0.5 mM ATP and 35 U of T4 polynucleotide kinase (Boehringer) for 30 min at 37°C.

0.67 μg of kinased Xhol linkers and 0.4 μg of Bal31 treated blunt end DNA of plasmid p31R are ligated overnight at room temperature in 25 μl of 60 mM Tris pH 7.5, 10 mM MgCl₂ 5 mM DTT, 3.5 mM ATP and 450 U of T4 DNA ligase. The ligated DNA is separated from excess linkers by isopropanol precipitation in the presence of 10 mM EDTA, 0.3 M sodiumacetate pH 6.0 and 0.54 volumes of isopropanol. After 35 min. of incubation at room temperature the DNA is sedimented by centrifugation. The pellet is dried at the air and resuspended in 17 μl of 6 mM Tris pH 7.9, 150 mM NaCl, 6 mM MgCl₂ and 6 mM mercaptoethanol. The Xhol linkers ligated to the DNA are cleaved with Xhol, the DNA is precipitated with isopropanol as described before and circularized by ligation. After 6 hours of ligation at 15°C in 50 μl of 60 mM Tris-ΗCl pH 7.5, 10 mM MgCl₂, 10 mM DTT, 1 mM ATP and 600 U of T4 DNA ligase, 10 μl of each ligation mixture are added to 100 μl of calcium-treated, transformation competent E. coli HBlOl cells (see Example Aa).

24 amp^R colonies are grown individually in LB medium containing 100 mg/l ampicillin. Plasmid DNA [p31R(XhoD] is isolated and analysed by Haelll digestion. Two plasmids are chosen and the nucleotide sequence adjacent to the Xhol site in the terminator fragment is determined by the technique of Maxam and Gilbert (12).

d) Transfer of the PHO5-TPA hybrid gene to the yeast vector pJDB207 (see fig. 12)

A RF plasmid preparation of M13mp9/PHO5-TPA is performed as described above. 2 μg of this DNA is digested with restriction endonuclease BamHI (Biolabs) and Sail (Biolabs) and the 2.6 kb fragment is isolated by electrophoresis on a low melting agarose gel (see Example Be). Likewise, 100 ng of the 134 bp Hindlll-Xhol fragment of plasmid p31R(XhoI) containing the PHO5 terminator are prepared. In addition, 1 μg of plasmid pJDB207 (29) is digested with BamHI and Hindlll and the 6.5 kb fragment is isolated by electrophoresis on a low melting agarose gel.

0.5 μg of each of the three fragments are ligated in 20 μl of 60 mM Tris.HCl, pH 7.5, 10 mM MgCl₂, 10 mM DTT, 1 mM ATP with 200 units of T₄ DNA ligase (Biolabs) overnight at 15°C. Transformation of E. coli HB 101 is performed as described in Example Aa, selecting for ampicillin resistant colonies. Four colonies are picked, plasmid DNA is prepared (Maniatis et al., Ref. 7, p. 92) and the DNA is analyzed using the following restriction sites as landmarks: BamHI, Hindlll, BstEII. All isolates are found to be correct. One of the plasmids is referred to as ρJDB207/PHO5-TPA(1A) . Example 8: In vivo ligation in S. cer. of rDNA with an insert DNA (Fig. 13)

The 2.1 kb Sail restriction fragment of the rDNA of Physarum polycephalum as described by Hattori et al. (34) is cloned into pBR322 using conventional methods. The obtained plasmid pBR322/rDNA 2.1 has a single Smal restriction site within the rDNA insert. Therefore this plasmid is cut by Smal and the 2.7 kb BamHI restriction fragment described in Fig. 2 containing the PH05 promoter, the TPA structural gene and the PHO5 terminator is inserted. In order to allow cloning of the BamHI fragment into the Smal site the recessed 3'ends of the BamHI site are filled using the Klenow fragment of E. coli polymerase I. The TPA gene is now flanked by rDNA sequences and is introduced into a yeast strain containing self-replicating rDNA molecules. In order to facilitate detection of the transformants the cotransformation technique of Hicks et al. (32) is used. The cotransforming plasmid is Yep 13 described by Broach et al. (33).

This method is also applicable for the in vivo construction of other rDNA/foreign gene hybrids which can be isolated from the transformed yeast strain and subsequently introduced into other cells, such as plant cells, or mammalian cells.

Example 9: Transformation of cells of Nicotiana tabacum c.v. Petit

Havana SRI with rDNA of P.p. For transformation the following samples were used: Sample 1: 10 μg of rDNA of P.p. + 10 μg of plasmid pABDl + 30 μg of calf thymus carrier DNA Sample 2: 10 μg of rDNA of P.p. + 40 μg of calf thymus carrier DNA Sample 3: 10 μg of rDNA of P.p. a) Construction of the pABDl plasmid (Fig. 14)

The freely available plasmids pKm 21 and pKm 244 [Beck, E. et al., Gene 19, 327-336 (1982)] are cut with the Pstl restriction endonuclease. The fragments of the plasmids which are used for recombination are purified by electrophoresis in 0.8 % agarose gel. The plasmid pKm 21244 resulting from the combination of the fragments contains a combination of the 5'- and 3'-Bal 31 deletions of the NPT II gene, as described by Beck et al. in Gene 19, 327-336 (1982). Joining the promoter signal of cauliflower mosaic virus to the Hindlll fragment of the plasmid pKm 21244 is effected by constructing the linker plasmid pJPAX. The coupling plasmid pJPAX is obtained from the plasmids pUC8 and pUC9 [Messing, J. and J. Vieira, Gene 119, 269-276 (1982)]. 10 base pairs of the linker sequence of the plasmid pUC9 are deleted by restriction at the Hindlll and Sall sites and the resultant cohesive ends are filled in by treatment with the polymerase I Klenow fragment [Jacobson, H. et al., Eur. J. Biochem. 45, 623, (1974)] and ligating the polynucleotide chain, thus restoring the Hindlll site. An 8 base pair synthetic Xhol linker is inserted at the Smal site of this deleted linker sequence. Recombination of the appropriate Xorl and Hindlll fragments of the plasmid pUC8 and of the modified plasmid pUC9 yields the plasmid pJPAX with a partially asymmetric linker sequence containing the following sequence of restriction sites: EcoRI, SMal, BamHI, Sail, Pstl, Hindlll, BamHI, Xhol and EcoRI. Joining of the 5' expression signals of the CaMV gene VI and the Hindlll fragment of the NPT II gene is carried out on the plasmid pJPAX by inserting the promoter region of the CaMV VI gene between the Pstl and Hindlll sites. The plasmid so obtained is restricted at its single Hindlll site and the Hindlll fragment of the plasmid pKm 21244 is inserted into this restriction site in both orientations, yielding the plasmids pJPAX CaKm⁺ and pJPAX CaKm - To provide an EcoRV site near the 3'-terminal region of the NPT II hybrid gene, a BamHI fragment of the plasmid pJPAX CaKm is inserted into the BamHI site of the plasmid pBR 327 [Soberon, X. et al., Gene 9, 287-305 (1980)], yielding the plasmid pBR 327 CaKm. The EcoRV fragment of the plasmid pBR 327 CaKm, which contains the new DNA construction, is used to replace the EcoRV region of the CaMV gene VI, which is cloned at the Sail site in the plasmid pUC8, thereby placing the protein-coding DNA sequence of the NPT II gene under the control of the 5' and 3' expression signals of the cauliflower mosaic gene VI. The plasmids so obtained are designated pABDl and pABDII respectively (q.v. Fig. 1).

b) Transformation of protoplasts of Nicotiana tabacum c.v. Petit

Havana SRI by transfer of the Sample 1 by PEG treatment Tobacco protoplasts at a population density of 2.10⁶ per ml are suspended in 1 ml of K₃ medium [q.v. Z. Pflanzenphysiologie 78, 453-455 (1976); Mutation Research 81 (1981) 165-175], containing 0.1 mg/l of 2,4-dichlorophenoxyacetic acid, 1.0 mg/l of 1-naphthylacetic acid and 0.2 mg/l of 6-benzylaminopurine, which protoplasts have been obtained beforehand from an enzyme suspension by flotation on 0.6 molar sucrose at pH 5.8 and subsequent sedimentation (100 g for 5 minutes) in 0,17 M calcium chloride at pH 5.8. To this suspension are added, in succession, 0.5 ml of 40 % polyethylene glycol (PEG) with a molecular weight of 6000 in modified (adjusted again to pH 5.8 after autoclaving) F-medium [Nature 296, 72-74 (1982)] and 65 μl of an aqueous solution containing Sample 1. This mixture is cultured for 30 minutes at 26°C with occasional agitation and subsequent stepwise dilution with F medium. The protoplasts are isolated by centrifuging (5 minutes at 100 g) and resuspended in 30 ml of fresh K₃ medium. Further incubation is carried out in 10 ml portions in Petri dishes of 10 cm diameter at 24°C and in the dark. The population density is 6.3.10⁴ protoplasts per ml. After 3 days the culture medium in each dish is diluted with 0.3 parts by volume of fresh K₃ medium and incubated for a further 4 days at 24°C and 3000 lux. After a total of 7 days, the clones developed from the protoplasts are embedded in a culture medium solidified with 1 % of agarose and containing 50mg/l of kanamycin, and cultured at 24°C in the dark by the bead type culture method [Plant Cell Reports, 2, 244-247 (1983)]. The culture medium is replaced every 5 days by fresh nutrient solution of the same kind. One kanamycin resistant clone was recovered and grown as a callus line and analysed for the presence of rDNA. c) Transformation of protoplasts of Nicotiana tabacum c.v. Petit Havana SRI by transfer of the Samples 2 and 3 by PEG treatment

The transformation was performed as described in Example 9b). The callus clones were grown without kanamycin selection pressure. Five clones from each treatment were randomly chosen for further analysis for the presence of rDNA.

d) Regeneration of tabacco plants

After 3 to 4 weeks of continued culturing in kanamycin-containing culture medium (Sample 1), the resistant calli of 2 to 3 mm diameter are transferred to agar-solidified LS culture medium [Physiol. Plant 18, 100-127 (1965)], containing 0.05 mg/ℓ of 2,4-dichlorophenoxyacetic acid, 2 mg/ℓ of 1-naphthylacetic acid, 0.1 mg/ℓ of 6-benzylaminopurine, 0.1 mg/ℓ of kinetin and 75 mg/ℓ of kanamycin. Kanamycinresistant Nicotiana tabacum Petit Havana SRI plants are obtained by inducing shoots on LS medium containing 150 mg/ℓ of kanamycin and 0.2 mg/ℓ of 6-benzylaminopurine, and subsequent rooting on T medium [Science 163, 85-87 (1969)].

The clones of samples 2 and 3 were regenerated without kanamycin.

e) DNA isolation from callus clones and leaf tissue Samples of 0.5 g of callus or leaf tissue were homogenised in a Dounce homogenizer in 3 ml of a buffer containing 15 % sucrose, 50 mM EDTA, 0.25 M NaCl, 50 mM Tris-HCl pH 8.0. Centrifugation of the homogenate for 5 min. at 1000 g resulted in a crude nuclear pellet which was resuspended in 2 ml of a buffer containing 15% sucrose, 50 mM EDTA, 50 mM Tris-HCl pH 8.0. SDS was added to a final concentration of 0.2% w/v. Samples were heated for 10 min at 70°. After cooling to room temperature, potassium acetate was added to a final concentration of 0.5 M. After incubation for 1 hour at 0° the precipitate formed was sedimented for 15 min in an Eppendorf centrifuge at 4°C. The DNA in the supernatant was precipitated with 2.5 volumes of ethanol at room temperature. DNA samples were further analysed for the presence of rDNA as described for S. cerevisiae in example 2. The results indicated the presence of rDNA of P.c.

In the same manner as described hereinbefore the rDNA containing a structural gene can be used for transformation of Nicotiana tabacum.

Description of the Figures 1-14

Fig. 1: Partial restriction map of the linear extrachromosomal rDNA of physarum polycephalum and ligation of the 2.7 kb PH05-TPA fragment flanked by BamHI restriction sites with BamHI/Bglll junctions to the fragments of the Bglll digested rDNA of Physarum polycephalum.

Fig. 2: Construction of 2.7 kb PHO5-TPA hybrid flanked by BamHI restriction sites.

Fig. 3: Starting plasmids pJDB207/PHO5,PHO3 and pBR322/PHO5Bam-Sal for DNA sequencing and plasmid construction.

Fig. 4: Construction of plasmid p30.

Fig. 5: Construction of expression plasmid 31.

Fig. 6: Nucleotide sequence of the Sau3A-PstI PHO5 transcription temriantion fragment.

Fig. 7: Deletion of the PHO5 signal sequence in expression plasmid p31.

Fig. 8: Collection of Bal31 deletions in the PHO5 region with EcoRI linkers at the arrows.

Fig. 9: Nucleotide sequence of the BamHI-EcoRI restriction fragment containing the PHO5R promoter region.

Fig. 10: Nucleotide sequence of the BamHI-EcoRI restriction fragment containing the PHO5Y promoter region.

Fig. 11: Construction of M13mp9/PHO5-TPA Fig. 12: Construction of the pJDB207/PHO5-TPA(la) plasmid, the starting plasmid used in Fig. 2

Fig. 13: Construction of a rDNA/TPA segment and in vivo insertion into rDNA of P.p.

Fig. 14: Construction of the plasmid pABDI containing the NPT-II structural gene

In the figures of the accompanying drawings, the symbols used have the following meanings:

deletion of a pBR 322 sequences restriction site yeast chromosomal DNA derived from PHO3, TPA gene PHO5 region yeast 2μ plasmid DNA deletion in pBR 322 yeast chromosomal DNA derived from LEU 2 restriction site

region amp^R ampicilline resistance gene direction of transcription tet^R tetracyclien resistance gene linker DNA stretch rDNA of Physarum polycephalum

Literature References

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3. Protozool. 27(1), 1980, 37-58

4. Daniel, JW. and Baldwin, H.H. (1964). Prescott, D.M., ed. Methods of Culture for Plasmodial Myxomycetes. Meth. Cell Physiol. Vol I, 9-41

5. Behrens, K. , Seebeck, T. and Braun, R. (1982). Aldrich, H.C. and Daniel, J.W., eds. Preparation of Ribomosal DNA. Cell Biology of Physarum and Didymium Vol II, 301-306

6. Beach and Nurse Nature 290, 140, (1981)

7. Molecular cloning. A laboratory Manual (eds. T. Maniatis, E.F. Fritsch, J. Sambrook), Cold Spring Harbor Lab. 1982, p. 177

8. B. Meyhack et al., EMBO Journal 1, 675 (1982)

9. M. Ranby, Biochim. Beiphys. Acta 704, 461 (1982)

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Claims

What is claimed is:

1. Eukaryotic hybrid vectors comprising an extrachromosomal rDNA or functional fragments thereof and a foreign structural gene coding for a polypeptide.

2. A eukaryotic hybrid vector according to claim 1, wherein the extrachromosomal rDNA is derived from protozoa of the two phyla Sacoromastigophera and Ciliophera, such as of the orders Dictyosteliida, Physarida, Tetrahymenina, Peniculina and Sporadotrichina.

3. A eukaryotic hybrid vector according to claim 1, wherein the extrachromosomal rDNA is derived from Dictyostelium discoideum, Tetrahymena species, Paramecium species, Oxytricha species, or Stylorichia species.

4. A eukaryotic hybrid vector according to claim 1, wherein the extrachromosomol rDNA is derived from Physarum polycephalum.

5. A eukaryotic hybrid vector according to claim 1, wherein functional fragments of the extrachromosomal rDNA of Physarum polycephalum obtained by digestion with Bgl II are used.

6. A eukaryotic hybrid vector according to claim 1, wherein the structural gene is coding for an enzyme.

7. A eukaryotic hybrid vector according to claim 1, wherein the structural gene is coding for a polypeptide hormone.

8. A eukaryotic hybrid vector according to claim 1, wherein the structural gene is coding for an immunomodulatory polypeptide.

9. A eukaryotic hybrid vector according to claim 1, wherein the structural gene is coding for an anti-viral or anti-tumor polypeptide.

10. A eukaryotic hybrid vector according to claim 1, wherein the structural gene is coding for an antibody.

11. A eukaryotic hybrid vector according to claim 1, wherein the structural gene is coding for a viral antigen.

12. A eukaryotic hybrid vector according to claim 1, wherein the structural gene is coding for a vaccine.

13. A eukaryotic hybrid vector according to claim 1, wherein the structural gene is coding for a clotting factor.

14. A eukaryotic hybrid vector according to claim 1, wherein the structural gene is coding for human TPA.

15. A eukaryotic hybrid vector according to claim 1, wherein the structural gene is coding for a polypeptide providing resistance against a plant pest.

16. A eukaryotic hybrid vector according to claim 1, wherein the structural gene is coding for a polypeptide providing resistance against excess temperature.

17. A eukaryotic hybrid vector according to claim 1 , wherein the structural gene is coding for a polypeptide providing resistance against dryness.

18. A eukaryotic hybrid vector according to claim 1, wherein the structural gene is coding for a polypeptide improving growth of the whole plant or parts thereof.

19. A eukaryotic hybrid vector according to claim 1, wherein the structural gene is linked to a promoter and/or other regulatory sequences.

20. A eukaryotic hybrid vector according to claim 1, wherein the structural gene is linked to the yeast PHO5 promoter.

21. A eukaryotic hybrid vector according to claim 1, comprising additionally a genetic marker squences.

22. Process for the preparation of a eukaryotic hybrid vector according to anyone of claims 1 to 21, characterized in that a foreign structural gene coding for a polypeptide and optionally additional DNA sequences are introduced in vitro or in vivo into an extrachromosomal rDNA or functional fragments thereof.

23. A eukaryotic cell transformed with a hybrid vector according to anyone of claims 1 to 22.

24. A transformed eukaryotic cell according to claim 23, which is selected from eukaryotic fungi.

25. A transformed eukaryotic cell according to claim 23, which is selected from yeasts, Aspergillus sp., Neurospora sp., Podospora sp., Penicillium sp., Cephalosporium sp., Mucor sp., especially Saccharomyces cerevisiae, Schizosaccharomyces pombe, Aspergillus niger, Neurospora crassa, Podospera anserina, Penicillium chrysogenum or Cephalosporium acremonium.

26. A transformed eukaryotic cell according to claim 23, which is selected from plant cells.

27. A transformed eukaryotic cell according to claim 23, which is selected from Solanaceae, Cruciferae, Compositae, Liliaceae, Vitaceae, Chenopodiaceae, Rutaceae, Bromeliaceae, Rubiaceae, Theaceae, Musaceae or Gramineae and of the order Leguminosae.

28. A transforemd eukaryotic cell according to claim 23, which is selected from families Graminea, Solanacea, Compositae, Cruciferae, and of the order Leguminosae.

29. A transformed eukaryotic cell according to claim 23, which is selected from invertebrates.

30. A transformed eukaryotic cell according to claim 23, which is selected from vertebrates.

31. A transformed eukaryotic cell according to claim 23, which is Saccharomyces cerevisiae .

32. A transformed eukaryotic cell according to claim 23, which is Nicotiana tabacum.

33. A method for producing transformed eukaryotic cells according to anyone of claims 23 to 30, characterized in that a transformable eukaryotic cell is treated with the eukaryotic hybrid vector under transforming conditions.

34. A method according to claim 33, characterized in that

a) for the transformation of a eukaryotic cell having a cell wall, the cell wall is removed and the obtained cell wall free spheroplast or protoplast is treated with the eukaryotic hybrid vector, e.g. in the presence of polyethyleneglycol (PEG) and Ca²⁺ ions, or

b) for the tranformation of eukaryotic cells without a cell wall, the eukaryotic cell is treated with the eukaryotic hybrid vector by calcium phosphate precipitation of the vector DNA onto the recipient cells, and,

if necessary regenerating the cell wall and selecting of or screening for the transformed cells.

35. A method for the preparation of a polypeptide, characterized in that eukaryotic cells according to anyone of claims 23 - 31 transformed with a eukaryotic hybrid vector coding for said polypeptide according to anyone of claims 1 to 21 are cultured, and, when required, said polypeptide is isolated.

36. The polypeptides whenever prepared according to the methods of claim 35.

37. The polypeptides obtainable according to claim 35.

38. The eukaryotic hybrid vectors, the transformed eukaryotic cells and the processes for their preparation as well as the method for producing the polypeptides as described in the Examples.

39. Use of extrachromosomal rDNA as a vector for introducing a foreign gene into eukaryotic cells.