EP1153132A1

EP1153132A1 - Nucleic acids that code for a nucleobase transporter

Info

Publication number: EP1153132A1
Application number: EP00907590A
Authority: EP
Inventors: Wolf B. Frommer; Bernd Gillissen; Lukas BÜRKLE; Bruno Andre
Original assignee: Individual
Current assignee: FROMMER, WOLF BERND
Priority date: 1999-02-19
Filing date: 2000-02-21
Publication date: 2001-11-14
Also published as: US7179954B1; WO2000049152A1; DE19907209A1; AU2912800A

Abstract

The invention relates to a nucleic acid that codes for a vegetable or animal nucleobase transporter. The invention also relates to the uses of said nucleic acid. The invention further relates to a fragment of nucleic acid, a construct containing said nucleic acid and/or a fragment thereof and a host cell with said nucleic acid, fragment and/or construct. The invention also relates to a method for producing a transgenic plant using the aforementioned nucleic acid and a method for influencing the nucleobase transport properties of a plant, part of a plant, a plant cell and/or seeds.

Description

Nucleic acids encoding nucleobase transporters

The present invention relates to a nucleic acid which codes for a plant or animal nucleobase transporter and to its use. The present invention further relates to a fragment of the nucleic acid, a construct which contains the nucleic acid and / or a fragment thereof, and a host cell. The present invention also includes a method for producing a transgenic plant and a method for influencing the nucleobase transport properties of a plant, a plant part, a plant cell and / or seeds.

Transporters play a special role in the function of an organism. On the one hand, they decide on the absorption or release of a substance into or from a cell or organism, on the other hand they control the transport and distribution of the substances between the cells. As a rule, transporters are at the beginning or end of a metabolic pathway and therefore generally perform superordinate control functions.

The transport metabolites that serve as building blocks for nucleic acids include purine bases, pyrimidine bases and the nucleosides and nucleotides derived from them. The uptake of these substances has, for example, an important physiological importance in the provision of precursors for nucleic acid synthesis during pollen germination and the early development of the embryo in germinating seeds. As phytohormones, cytokinins are structurally closely related to the purine bases and the purine nucleosides. These phytohormones regulate many processes during plant development. Little is known about the origin of these hormones, but their effective transport in the plant is of crucial importance for their development and function.

Nucleobase transpσrt systems for adenine, cytosine and uracil were characterized in bacteria and the corresponding genes could be cloned. In the baker's yeast Saccharomyces cerevisiae, three different, active transport systems for nucleosides and nucleobases have so far been characterized both genetically and physiologically. Nucleoside transporter systems have been described in a variety of mammalian cells In addition to these nucleoside transporter systems, specific transport systems for nucleobases in mammalian cells have also been described

In higher plants, transport processes for the distribution of assimilates, metabolites and phytohormones are of crucial physiological importance.However, very little is known about the transport of nucleobases and their derivatives in plants and, in contrast to bacteria, fungi and mammals, only a few transport systems have so far been known for them Substances described A clarification of the nucleobase transport processes in plants, which would lead to similarly detailed information as in other organisms, is not available and is hardly feasible due to the difficulty of the molecular biological analysis of mutations in plants. However, there is great interest in the Identification and characterization of plant genes which code for nucleobase transporters or for transporters for chemically related substances. Furthermore, due to the central function of the transporters, there is great interest in plants which are able to produce larger amounts of nu to transport kleobases and their derivatives, and to provide opportunities for changing the distribution of nucleobases in transgenic plants and mutants

The object of the present invention is to provide nucleic acids coding for plant or animal nucleobase transporters

According to the invention, this object is achieved by a nucleic acid selected from

a) nucleic acid which can be obtained by complementing host cells deficient in nucleobase transporter with a plant or animal gene bank and selecting host cells positive for nucleobase transporters,

b) nucleic acid with a sequence which codes for a protein with a sequence according to SEQ ID NO 8 or SEQ ID NO 9,

c) nucleic acid which hybridizes with a nucleic acid according to b), d) nucleic acid which, taking into account the degeneration of the genetic code, would hybridize with a nucleic acid according to b) or with the sequence complementary to b);

e) derivatives of a nucleic acid obtained by substitution, addition, inversion and / or deletion of one or more bases according to a) to d);

f) complementary nucleic acid to a nucleic acid according to one of groups a) to e);

except for nucleic acids with a sequence according to one of SEQ ID NO 3 to 5.

The term "nucleic acid base" as used here includes not only nucleobases and their derivatives, but also substances chemically related to the nucleobases, for example adenine, guanine and its derivatives, such as xanthine, hypoxanthine, allantoin, allantoate, urate, xanthosine or inosine, Cytosine and its precursors and derivatives such as barbiturates or folic acid, cytokinins such as Zeatin, isopentenyladenine or kinetin, and certain alkaloids such as e.g. Caffeine, theobromine or nicotine. These vegetable alkaloids show a great structural similarity to the nucleic acid purine because they also have basic, N-containing heterocycles. Cytokinins contain adenine as a hydrophilic backbone, on the amino group of which there is a non-polar side chain of relatively low specificity in position 6. The kinetin is an artificial cytokinin, which probably does not occur naturally in the plant. Furthermore, the nucleobases can be modified and coupled to sugar or other building blocks, e.g. Adenosine as a riboside of adenine, cytidine as a riboside of cytosine, or cytokinin riboside.

The term “nucleobase transporter” in the sense of the invention denotes a protein which is involved in the transport of at least one of the aforementioned metabolites through a biomembrane. This transport can take place actively or passively. To detect the nucleotransporter activity, for example, that of Ninnemann et al. (1994, EMBO J. 15, 3464-3471) can be used.

"Comple entation", as used here, denotes a compensation of a genetic functional defect that is reflected in the phenotype while maintaining the same Defective underlying mutations. Complementation within the meaning of the invention is present, for example, when a genetic defect in a gene (for example the FCY2 gene in Saccharomyces cerevisiae) is eliminated by the presence of a similar, intact gene (for example the PUP1 gene from Arabidopsis thaliana) which has the function of the defective gene takes over.

"Nucleobase transporter-deficient host cells" in the sense of the invention means cells which, because of a genetic defect, have negatively changed nucleobase transport properties which lead to a negatively selectable phenotype. Preferred nucleobase transporter deficient host cells for carrying out the invention are eukaryotic cells, e.g. plant or animal cells. Nucleobase transporter-deficient yeast cells are particularly preferred.

Nucleobase transporter-positive host cells contain a nucleic acid that at least partially eliminates the genetic defect and therefore show a positively selectable phenotype.

A nucleobase transporter can be identified, for example, by complementing specific mutations in the yeast Saccharomyces cerevisiae. To isolate a gene encoding a transporter molecule from a plant or animal gene bank, suitable yeast mutants must first be available, which are not able to take up a specific substance due to a defect in this transporter molecule. A mutant that is not able to grow in media with nucleobases as the sole nitrogen source is, for example, that of Grenons (Grenson, 1969, Eur. J. Biochem. 11, 249-260; Polak & Grenson, 1973, Eur. J. Biochem. 32, 276-282) described mutant fcy2 (strain MG887). In order to be able to carry out a complementation with plant or animal genes, the URA3 gene was additionally destroyed and thus a uracil auxotrophy was generated (MG887ura3 -) -

To obtain a nucleic acid according to the invention, a suitable yeast mutant, such as, for example, the fcy2 / ura3 mutant, can be used with expression plasmids suitable for use in yeast, which insert cDNA fragments from a plant or animal cDNA library, be transformed. Plant or animal nucleobase transporters are identified by selecting transformants which are able to grow on nucleobases as the only nitrogen source as a result of the expression of plant or animal cDNA sequences.

It has now surprisingly been found that when a cDNA library is expressed, for example from Arabidopsis thaliana seedling tissue, by means of expression plasmids which contain the yeast phosphoglycerate kinase promoter and are suitable for use in yeast, the mutation fcy2 can be complemented if possible the expression plasmids contain certain plant cDNA fragments. These cDNA fragments encode plant nucleobase transporters and are encompassed by the present invention.

The feature “nucleic acid that hybridizes with a nucleic acid according to b)”, as used here, indicates a nucleic acid that hybridizes with the nucleic acid according to b) under moderately stringent conditions. For example, the hybridization with the radioactive gene sample in a hybridization solution (25% formamide; 5 x SSPE; 0.1% SDS; 5 x Denhardt's solution; 50 μg / ml herring sperm DNA; for the composition of the individual components see Sambrook et al. , 1989, Molecular Cloning: A laboratory manual, 2nd edition, Cold Spring Harbor Laboratory Press, NY, USA) for 20 hours at 37 ° C. The subsequent removal of unspecifically bound samples can be done by washing the filters several times in 2 x SSC / 0.1% SDS at 42 ° C. The filters are preferably washed with 0.5 x SSC / 0.1% SDS, particularly preferably with 0.1 x SSC / 0.1% SDS at 42 ° C.

The nucleic acids according to the invention can be introduced into plasmids and subjected to mutagenesis or a sequence change by recombination using standard microbiology methods. This allows a particularly simple change in the specificity of the nucleobase transporter. Nucleic acids that code for modified nucleobase transporters can be used, for example, for the transformation of agricultural plants with the aim of producing transgenic plants. Using standard procedures (see Sambrook et al., 1989, Molecular cloning: A laboratory manual, 2nd edition, Cold Spring Harbor Laboratory Press, NY, USA) base exchanges can be made or natural or synthetic sequences can be added. To connect fragments to one another, adapters or "linkers" can be added to the fragments. Further mani may populations, ^~ provide suitable Restriktioπsschnittstellen or redundant sequences, or restriction sites remove, can be used. In order to make insertions, deletions or substitutions, such as, for example, transitions and transversions, known methods are used, such as, for example, in vrtra mutagenesis, "primer repair" restriction or ligation. General methods, such as sequence or restriction analysis, as well as other biochemical-molecular biological methods are used to analyze the nucleic acid according to the invention.

A nucleic acid encoding a polypeptide or protein with nucleobase transporter activity which has at least 40%, preferably at least 60%, particularly preferably at least 80% homology to one of the nucleic acid according to SEQ ID NO 1 or the nucleic acid according to SEQ ID over its entire sequence Having NO 10 encoded polypeptide is also encompassed by the present invention.

For the purposes of the invention, the expression "at least 40%, preferably at least 60%, particularly preferably at least 80% homology" refers to agreement at the level of the amino acid sequence, which according to known methods, e.g. of computer-aided sequence comparisons (Basic local alignment search tool, S.F. Altschul et al., J. Mol. Biol. 215 (1990), 403-410).

The term "homology" known to those skilled in the art denotes the degree of relationship between two or more polypeptide molecules, which is determined by the agreement between the sequences, whereby agreement means both identical agreement and conservative amino acid exchange. The percentage of "Homology" is the percentage of areas that match in two or more sequences, taking gaps or other sequence peculiarities into account.

The term "conservative amino acid exchange" refers to an exchange of one amino acid residue for another amino acid residue, the exchange does not change polarity or charge. An example of a conservative amino acid exchange is the replacement of a non-polar amino acid residue by another non-polar amino acid residue. Conservative amino acid exchanges in the sense of this invention are: G = A = S, I = V = L = M, D = E, N = Q, K = R, Y = F, S = T, G = A = I = V = L = M = Y = F = W = P = S = T.

The homology of related polypeptide molecules can be determined using known methods. As a rule, special computer programs with algorithms that take account of the special requirements are used. Preferred methods for determining homology initially produce the greatest agreement between the sequences examined. Computer programs for determining homology between two sequences include, but are not limited to, the GCG program package, including GAP (Devereux, J., et al., Nucleic Acids Research 12 (12): 387 (1984); Genetics Computer Group University of Wisconsin, Madison, (Wl)); BLASTP, BLASTN and FASTA (Altschul, S. et al., J. Molec Biol 215: 403/410 (1990)). The BLAST X program can be obtained from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Handbook, Altschul S., et al., NCB NLM NIH Bethesda MD 20894; Altschul, S., et al., J. Mol. 215: 403/410 (1990)). The well-known Smith Waterman algorithm can also be used to determine homology.

Preferred parameters for sequence comparison include the following:

Algorithm: Needleman and Wunsch, J. Mol. Biol 48: 443-453 (1970)

Comparison matrix: BLOSUM 62 by Henikoff and Henikoff, Proc. Natl. Acad.

Be. USA 89: 10915-10919 (1992)

Gap Penalty: 12

Gap length value

(Gap Length Penalty): 4

Similarity Threshold: 0

The GAP program is also suitable for use with the above parameters. The above parameters are the default parameters for amino acid sequence comparisons. Further exemplary algorithms, gap opening penalties, gap extension penalties, comparison matrices including those mentioned in the program manual, Wisconsin package, version 9, September 1997, can be used. The selection is made by depend on the comparison to be carried out and also on whether the comparison is carried out between pairs of sequences, GAP or Best Fit being preferred, or between a sequence and an extensive sequence database, with FASTA or BLAST being preferred

The invention also encompasses a nucleic acid which encodes a polypeptide or protein with nucleobase transporter activity which, over a partial range of at least 20 amino acids, has at least 60%, preferably at least 75%, particularly preferably at least 90% homology to one of those of the nucleic acid SEQ ID NO 1 and the nucleic acid encoded according to SEQ ID NO 10 comprises preferably the nucleic acid sequence according to the invention encoding a polypeptide or protein with nucleobase transporter activity which comprises an amino acid sequence which has at least 70%, preferably at least 80%, particularly preferably at least 90% homology to one of the following amino acid sequences

(a) LYAXGLXYLPVSTXSLIXXXQLAFXAXFS (SEQ ID NO 11)

where X at positions 4 and 7 for a hydrophobic amino acid (G, A, I, V, L, M, Y, F, W, P, S or T), at positions 14 and 18-20 for any amino acid, at position 25 is T or N and at position 27 is I or F,

(b) LGXVGLIFXXSSLFSXVXXXXXLPV (SEQ ID NO 12)

where X at positions 3 and 18-22 for a hydrophobic amino acid (G, A, I, V, L, M, Y, F, W, P, S or T), at position 10 for any amino acid position 9 is L or E and position 16 is G or N,

(c) LLLXIWGFXSYXYX (SEQ ID NO 13) where X at positions 9 and 12 for a hydrophobic amino acid (G, A, I, V, L, M, Y, F, W, P, S or T), at position 4 for S or A and at the position 14 represents Q or S.

The nucleic acid preferably comprises the coding sequence of one of the sequences according to SEQ ID NO 1, 2, 6, 7 or 10, or a derivative derived therefrom by substitution, addition, inversion and / or deletion of one or more bases. In a particularly preferred embodiment of the invention, the nucleic acid is a DNA.

The invention also relates to a fragment of the nucleic acid according to the invention which, in the antisense orientation to a promoter, can inhibit the expression of a nucleobase transporter in a host cell. This fragment can comprise at least 10 nucleotides, preferably at least 50 nucleotides, very particularly preferably at least 200 nucleotides. This fragment can be introduced into a host cell and transcribed there into a non-translatable RNA (Antisinπ-RNA), which can inhibit its expression by binding to an endogenous nucleobase transporter gene or to the mRNA transcribed therefrom.

The invention further relates to a construct which contains a nucleic acid according to the invention and / or a fragment thereof according to the invention under the control of the elements regulating the expression. Examples of such regulating elements are constitutive or inducible promoters, for example the bacteria E. coli promoter araBAD (Carra & Schlief, 1993, EMBO J. 12, 35-44), for fungi the yeast promoter PMA1 (Rentsch et al., 1995, FEBS Lett. 370, 264-268) and for plants the viral CaMV35S promoter (Pietrzak et al., 1986, Nucl. Acids Res. 14, 5857-5868). Furthermore, the nucleic acid or the fragment can be provided with a transcription termination signal. Such elements have already been described (see, for example, Gielen et al., 1984, EMBO J. 8, 23-29). The transcriptional start area can be both native (homologous) and foreign (heterologous) to the host organism. The sequence of the transcription start and termination regions can be produced synthetically or obtained naturally or contain a mixture of synthetic and natural components. The nucleic acid or the fragment in the construct is preferably in an antisense orientation to the regulatory element. The construct can for example, are introduced into the plant genome and, after its transcription, lead to the suppression of the formation of the plant's own nucleobase transporter molecules. In a particularly preferred embodiment of the invention, the construct is present in a plasmid.

The plasmid can contain a replication signal for E. coli or yeast and a marker gene which allows positive selection of the host cells transformed with the plasmid. If the plasmid is introduced into a plant host cell, depending on the introduction method, further sequences may be required, which are known to the person skilled in the art. If the plasmid according to the invention is, for example, a derivative of the Ti or Ri plasmid, the nucleic acid or the fragment to be introduced must be flanked by T-DNA sequences which enable the integration of the nucleic acid or the fragment into the plant genome. The use of T-DNA for the transformation of plant cells has been intensively investigated and i.a. in EP 120 516, Hoekema, The Binary Plant Vector System, Offset-drukkerij Kanters B.V. Ablasserdam, (1985) Chapter V, Fraley et al., Crit. Rev. Plan Be. 4, 1-46 and An et al., 1985, EMBO J. 4, 277-287. Once the nucleic acid or fragment introduced has been integrated into the genome, it is generally stable there and is also retained in the offspring of the originally transformed cell. The integrated sequence can also contain a selection marker which gives the transformed plant cells resistance to a biocide or an antibiotic, e.g. Kanamycin, G418, bleomycin, hygromycin or phosphinotricin mediated. The individually used marker should therefore allow the selection of transformed cells against cells that lack the inserted DNA.

The invention further relates to a host cell which contains a nucleic acid according to the invention and / or a nucleic acid with a sequence according to one of SEQ ID NO 3 to 5 and / or a fragment of the aforementioned nucleic acids and / or a construct according to the invention. The host cell according to the present invention can be selected from bacteria, yeast, mammalian and plant cells.

Furthermore, the present invention relates to a transgenic plant and to plant parts and / or seeds of this plant which contain a nucleic acid according to the invention or a Contain nucleic acid with a sequence according to one of SEQ ID NO 3 to 5 and / or a fragment of the aforementioned nucleic acids and / or a construct according to the invention. Preferably, the nucleic acid, fragment and / or construct is integrated at a genome site that does not correspond to its natural position.

The present invention also relates to a protein which can be obtained in a host cell by expression of a nucleic acid according to the invention or a nucleic acid with a sequence according to one of SEQ ID NO 3 to 5. The respective initiation codons (ATG) of the sequence numbers 1 to 7 were identified in the sequence listing by underlining. The protein preferably has the same nucleobase transport properties as the protein encoded by SEQ ID NO 1 or SEQ ID NO 9. To detect the activity of such a protein, for example, absorption experiments can be carried out, as described in the exemplary embodiment. Antibodies that react with a protein according to the invention are also included in the invention.

Another object of the invention is to provide a method for producing a transgenic plant. This problem is solved by a method which comprises the following steps:

Introducing a nucleic acid according to the invention or a nucleic acid with a sequence according to one of SEQ ID NO 3 to 5 and / or a fragment according to the invention of the aforementioned nucleic acids into a plant cell; and

- Regeneration of a plant from the transformed plant cell.

Transgenic plants which are produced according to the method according to the invention can e.g. derived from tobacco, potato, tomato, sugar beet, soybean, coffee, pea, bean, cotton, rice or corn plants.

In addition to the transformation with the aid of agrobacteria, numerous other techniques are available for introducing the nucleic acid or the fragment into a plant cell. These techniques include protoplast fusion, micro-injection of DNA, electroporation, and ballistic methods and virus infection. Whole plants can then be regenerated from the transformed plant cells in a suitable medium, which can contain antibiotics or biocides for selection. The plants thus obtained can then be tested for the presence of the introduced DNA.

In contrast to the transformation using agrobacteria, there are no special requirements for the vector in the case of injection and electroporation. Simple plasmids such as e.g. pUC derivatives can be used. However, if whole plants are to be regenerated from such transformed cells, the presence of a selectable marker gene is advantageous. The transformed cells grow within the plants in the usual way (see also McCormick et al., 1986, Plant Cell Reports 5, 81-84). These plants can be grown as usual and crossed with plants that have the same transformed genetic makeup or other genetic makeup. The resulting hybrid individuals have the corresponding phenotypic properties.

The present invention also relates to a method for influencing the nucleobase transporter properties of a plant, a plant part, a plant cell and / or seeds, which comprises the following step:

- Introducing a nucleic acid according to the invention or a nucleic acid with a sequence according to one of SEQ ID NO 3 to 5 and / or a fragment according to the invention of the aforementioned nucleic acids into a plant cell and / or a plant.

Both changes in the specificity of the transport system which enable the transport of new compounds and those which cause a change in the transport mechanism are suitable for influencing the nucleobase transport properties of a plant. For example, changes are possible that change the affinity or substrate specificity of the transporter such that there is a more efficient nucleobase transport into the leaves or a change in apical dominance, flowering behavior or senescence, or that an improved distribution of pesticides is made possible. The plant cells according to the invention can be used for the regeneration and production of whole plants. The nucleic acid according to the invention and nucleic acids with a sequence according to one of SEQ ID NO 3 to 5 can be used to isolate homologous sequences from bacteria, fungi, plants, animals and / or humans. In order to be able to search for homologous sequences, gene banks must first be created which are representative of the content of genes of an organism or of the expression of genes in this organism. The former are genomic, the latter are cDNA libraries. From these, sequences can be isolated from the aforementioned nucleic acids using a probe. Once the associated genes have been identified and isolated, the sequence can be determined and the properties of the proteins encoded by this sequence can be analyzed.

Another use of the aforementioned nucleic acids relates to the expression of a nucleobase transporter in pro- and / or eukaryotic cells. If the aforementioned nucleic acids are introduced into a prokaryotic cell, an RNA sequence of a eukaryotic nucleobase transporter that can be translated by bacteria is formed, which despite the considerable differences in the membrane structures of pro and eukaryotes is translated into a functional eukaryotic nucleobase transporter with its substrate specificity. This enables the use of bacterial strains for studies of the properties of the transporter and its substrates. The aforementioned nucleic acids can also be used under the control of a regulatory element in an antisense orientation to inhibit the expression of an endogenous nucleobase transporter in pro- and / or eukaryotic cells. The production of transgenic crop plants is another possible use of these nucleic acids.

Other uses are:

• If the transporters are essential for the function of the plant, they can serve as a herbicide target: screening methods in yeast to look for inhibitors, these can be optimized in the yeast system and by chemical modification and then tested on plants. • Since substitutions on the substrate framework are permitted, the transporters can be used to mobilize pesticides, for example by attaching purine or pyrimidine residues to a fungicide or insecticide.

Special uses are:

1.Substrate class A: transporters are responsible for the transport of secondary metabolites or alkaioids such as Caffeine, theobromine, nicotine and similar substances.

• Overexpression or ectopic expression under the control of various promoters e.g. CaMV-35S or specific promoters for secondary metabolites of this substance class better in certain organs, e.g. Transport leaves for harvest, seeds and tubers or beets.

Cosuppression or antisense repression around the content of such secondary metabolites, especially toxic substances in certain organs, e.g. Reduce food products; e.g. as a new decaffeination process.

• Secondary metabolites are important plant defenses against infections and animal feed. Improved care for affected organs can lead to improved resistance and resistance.

• Secondary metabolites as pharmaceutical products (plant as a bioreactor). An optimal supply of harvest organs is essential for extractability and can be achieved through optimized transport in transgenic plants.

2. Substrate class B: transporters are responsible for the transport of nucleobases and derivatives, eg on the one hand adenine, xanthine, allantoin, allantoat, hypoxanthine, urate, xanthosine, inosine; on the other hand, cytosine and its derivatives such as barbiturates and folic acid, but also nucleosides such as adenosine etc., lead to reversal of transport processes in transgenic plants • changed cell division activity and improved development of autonomous cells: nucleobases play an important role in DNA and RNA synthesis and thus cell division activity, for example pollen is supplied with nucleobases. Use for the production of male sterile pollen by transport inhibition;

• in the transport of allantoic acid a role in the fixation of atmospheric nitrogen. Intercellular transport is important for the nitrogen assimilation of the plant.

• Adenine is an essential building block of ATP. The external supply e.g. of the phloem with ATP could be influenced positively or negatively in transgenic plants by changing the expression of PuP transporters.

3.Substrate class C: transporters are responsible for the transport of plant hormones, e.g. Cytokinins and cytokine derivatives, e.g. Ribosides (see adenine / adenosine).

Since cytokinins play a central role in the control of developmental and metabolic processes, a change in activity can be caused by

• Overexpression, ectopic expression or repression (through cosuppression or antisense) in transgenic plants for improved yields (control of sink-source relations), changed degree of branching (role in apical dominance), improvement of the germination behavior of seeds, acceleration or deceleration of bud formation and acceleration or deceleration of senescence (cytokinins slow senescence). In addition, cell division activity and thus the formation of side roots, the size and capacity of yielding organs, the morphogenesis in tissue culture, the expansion of leaves and the regulation of water efficiency are influenced due to the influence on stoma opening and plastid development.

4. Transport of related substances: Auxins are structurally close to the spectrum of substrates mediated by nucleobase transporters and could can also be conveyed by the transporters. If the transport of auxins is changed, the entire spectrum of auxin effects can be influenced.

5. Modification of the transport properties of the transporters by mutagenesis: expansion of the substrate spectrum, e.g. towards the transport of alkaloids that have not yet been recognized by the transporters, synthetic hormones that are poorly transported, nucleobases that are not transported efficiently enough as a starting point for changing transport processes in transgenic plants.

So far, homologs have been found for almost all new plant transporters in animal / human genomes (examples: glucose transporters of the plant MST family are related to animal GLUT transporters at the sequence level; amino acid transporters of the AAP family were later found in animals (VGAT, SN1) ; Amino acid transporters of the CAT family in animals and plants are related). Based on the similarity of the biochemical properties found for the animal / human transport of adenine and the competition by caffeine, it is postulated that a previously unidentified homologue of the PUP family in these systems is responsible for the nucleobases / nucleoside and derivative transport is. The expression of this system in yeast cells for functional verification enables firstly the description, secondly the identification of possible hereditary diseases that are caused by defects in the system, the identification of new lead structures for pharmaceuticals and the influencing of the transport of substances via the blood / brain barrier, because the adenine / caffeine transport occurs at this point.

The following figures and examples illustrate the invention.

FIG. 1 shows a hydrophobicity analysis of the PUP nucleobase transporter protein according to Kyte and Doolittle.

FIG. 2 shows the results of an uptake experiment shown in the form of a diagram, in which the cytosine uptake rate of the yeast strain MG877ura3 ^_ :: pFL61-PUP1 and the wild-type strain ∑1278b (Dubois & Grenson, 1979, Mol. Gen. Genet. 175, 67-76) was measured at different pH values. The substrate concentration was 100 μM.

FIG. 3 shows the results, shown in the form of a diagram, of an uptake experiment in which the cytosine uptake of the yeast strain MG877ura3 ^~ :: pFL61-PUP1 was measured with and without the addition of glucose. The cells were washed twice with water before starting the uptake measurement and incubated for 30 minutes at room temperature. Five minutes after the start of the measurements, glucose was added to one of the test batches to a final concentration of 1%.

FIG. 4 shows an analysis of the substrate specificity of the PUP1 nucleobase transporter (black bars) expressed in yeast in comparison to the yeast-specific FCY2 transporter (white bars).

FIG. 5 shows the results of a recording experiment shown in the form of a diagram, in which the cytokinin uptake of the yeast strain MG877ura3 ^- :: PUP1 was investigated by means of ³ H-labeled zeatin. The uptake per time at a concentration of 100 μM trans-zeatin is indicated. In this case, PUP1 was expressed under the control of the yeast ATPase promoter PMA1 in the vector pDR195 (Rentsch et al., 1995, FEBS Lett. 370, 264-368) in the mutant MG877ura3 ^~ (FCY2). The empty vector (FCY2pDR195) served as a control.

FIG. 6a shows a schematic representation of the plasmid p35S-PUP1, a derivative of the plasmid pBIN19 (Bevan, 1984, Nucl. Acids Res. 12, 8711-8721). A stands for a fragment from the genome of the cauliflower mosaic virus, which carries the 35S promoter (nt 6909-7437). The promoter fragment was prepared as an Eco /? // K n / fragment from the plasmid pDH51 (Pietrzak et al., Nucl. Acids Res. 14, 5857-5868). B stands for a / Votf / Λ / otf fragment of the cDNA, flanked by a polylinker region from pT7T3 18U // vof / with Kpnl and Xöa / interfaces with the coding region of the nucleobase transporter of Arabidopsis thaliana in the orientation of fragment A. Der in The arrow drawn in fragment B indicates the reading direction of the cDNA. C stands for a polyadenylation signal of gene 3 of the T-DNA of the plasmid pTiACHδ (Gielen et al., 1984 EMBO J. 3, 835-846), nucleotides 11749 to 11939, which is isolated as a Pvull / Hindlll fragment from the plasmid pAGV40 (Herrera-Estrella et al., 1983, Nature 303, 209-213) and after Addition of a SpΛ / linker to the Pvu // interface between the Sphl and the H / nα7 // interface of the polylinker from pBIN19 was cloned.

FIG. 6b shows a schematic representation of the plasmid p35S-α-PUP1, a derivative of the plasmid pBIN19 (Bevan, 1984, Nucl. Acids Res. 12, 8711-8721). A and C each represent the CaMV35S promoter and the polyadenylation signal of gene 3 of the T-DNA of the plasmid pTiACHδ (see FIG. 6a). B stands for an Ssp /// - / pa / fragment of the cDNA with the coding region of the nucleobase transporter from Arabidopsis thiana in an antisense orientation to fragment A. The arrow drawn in fragment B indicates the reading direction of the cDNA.

FIG. 7 shows the measurement of inward currents in enopus oocytes which express PUP1 at a holding voltage of -80mV. Inward currents are measured as soon as adenine is added. The current intensity depends on the concentration (bars indicate the current on the ordinate and the time on the abscissa).

FIG. 8 shows: (A) the growth of DM734-284DpDR195 on 150 μM adenosine (control) and (B) the growth of DM734-284DpDR195PuP1 on 150 μM adenosine.

FIG. 9 shows the growth of Saccharomyces cerevisiae MG877ura3 ^~ (K) and clones transformed with the construct pDR195PUP1 (1) or with the construct pDR195PUP2 on caffeine plates with different caffeine concentrations.

EXAMPLES

General methods

a) Cloning method: The vector pT7T3 18U (Pharmacia) was used for cloning in E. coli and the vector pFL61 (Minet & Lacroute, 1990, Curr. Gene..18, 287-291) for yeast transformation. The genetic constructions cloned into the binary vector pBinAR, a derivative of pBIN19 (Bevan, 1984, Nucl. Acids Res. 12, 8711-8721).

b) Bacterial and yeast strains: The E. co // strain DH5α was used for the pT7T3 18U and pFL61 vectors and for the pBinAR constructs. The yeast strain MG887 (Dubois & Grenson, 1979, Mol. Gen. Genet. 175, 67-76) with the mutation fcy2 was used as the starting strain for the expression of the cDNA library in yeast after an ura3 deficiency had been introduced.

c) Transformation of Agrobactehum tumefaciens: The DNA transfer to the agrobacteria was carried out by direct transformation according to the Höfgen and Willmitzer method (1988, Nucl. Acids Res. 16, 9877). The plasmid DNA of transformed agrobacteria was isolated by the method of Bimboim and Doly (1979, Nucl. Acids Res. 7, 1513-1523) and analyzed by gel electrophoresis after suitable restriction cleavage.

d) Plant transformation: The plant transformations can be carried out by gene transfer mediated by Agrobactehum tumefaciens (strain C58C1, pGV2260) (Deblaere et al., 1985, Nucl. Acids Res. 13, 4777-4788). The transformation of A. thaliana is carried out, for example, by means of vacuum infiltration (modified from Bechtold et al. (1993) Comptes Rendus de l'Academie des Sciences Serie III, Sciences de la Vie 316: 1194-1199). Pots (diameter 10 cm) are filled with earth and then covered with a fly net. A. thaliana seeds are sown on this net. Six to eight weeks after sowing, the plants were used for vacuum infiltration. For the vacuum infiltration, 2 x 1 liter cultures in YEB + antibiotic (50 μg / ml Kan and

100 μg / ml Rif) at 28 ° C. With an OD ₆₀₀ of 1.5, the cells are harvested at 3000 g and resuspended in 600 ml of infiltration medium (0.5 × MS medium (Sigma), 5% sucrose, 44 μM benzylaminopurine). The bacterial suspension is filled into 250 ml mason jars and placed in a desiccator. The A. thaliana plants are immersed "upside down" in the bacterial suspension, then a vacuum is applied for 5 min. After 3-4 weeks, the seeds of these plants are harvested. For surface sterilization, the seeds are shaken in 4% sodium hypochloride, 0.02% triton for 10 min, centrifuged at 1500 g, washed four times with sterile water and resuspended in 3 ml 0.05% agarose per 5000 seeds. The Sameπ-agarose solution is spread on MSS medium (1 x MS, 1% sucrose, 0.8% agarose, 50 μg / ml kamanycin, pH 5.8) (plates with 13.5 cm diameter for 5000 seeds ). To reduce moisture loss, the panels are sealed with Parafilm ^® . The kanamycin-resistant plants are transferred to soil. Seeds from these plants are harvested and analyzed.

e) Detection of the nucleobase transporter activity: The activity detection can be carried out, for example, by taking up experiments with radioactive substrates (eg [ ¹⁴ C] adenine) in the cy2 yeast mutants transformed with the plant transporter gene under the control of the yeast promoter (MG877ura3 ^~ :: pFL61-PUP1), in principle as described in Ninnemann et al., 1994 (EMBO J. 15, 3464-3471). Alternatively, other expression systems, for example Xenopus oocytes (Borer et al., 1996, J. Biol. Chem. 271, 2213-2220), can be used using electrophysiological measurement methods.

Example 1: Cloning of the PUP1 nucleobase transporter gene from Arabidoosis thaliana

The PUP1 nucleobase transporter is cloned by complementing the yeast strain MG887ura3 ~ (fcy2) (this work; precursor MG887 Grenson 1969, Eur. J. Biochem. 11, 249-260) with a cDNA library from Arabidopsis thaliana and selecting the Nucleobase transporter positive cells. The fcy2 yeast mutant cannot grow in media with adenine or cytosine as the sole nitrogen source (Grenson, 1969, Eur. J. Biochem. 11, 249-260; Polak & Grenson, 1973, Eur. J. Biochem. 32, 276-282 ). To introduce an auxotrophy marker (ura3 ^~~ ), the nucleobase uptake-deficient mutant MG887 (Dubois & Grenson, 1979, Mol. Gen. Genet 175, 67-76) was transformed with a fragment of the URA3 gene, which carries an internal deletion. A URA3-deficient mutant MG887ura3 ~ was isolated by selection on the toxic precursor 5-fluoroorotate.

To complement the nucleobase transport mutation of the yeast strain MG887ura3 ^~ , the yeast expression vector pFL61 (Minet & Lacroute, 1990, Curr. Genet. 18, 287-291) cloned cDNA from young seedlings of Arabidopsis thaliana (two- Leaf stage) (Minet et al., 1992, Plant J. 2, 417-722). About 1 μg of the vector with a cDNA insertion was in the yeast strain MG887ura3 ^~ by the method of Dohmen et al. (1991, Yeast 7, 691-692). Yeast transformants that could grow on minimal medium with 1 mM adenine as the only nitrogen source were propagated. Plasmid DNA was isolated from these clones by standard methods. The strain MG887ura3 ^~ was transformed again with the isolated plasmid. In this way a plasmid pFL61-PUP1 was obtained which can complement the fcy2 mutation. This plasmid has an insert with a size of 1.2 kb which contains the PUP1 - nuclear base transporter gene.

The yeast strain MG887ura3 ^~ :: pFL61-PUP1 obtained by transforming MG887ura3 ^~ with the plasmid pFL61-PUP1 is used for uptake studies with adenine or nucleobases. By genetic modification of the coding region of the nucleus transporter gene PUP1 according to standard methods (cf. Sambrook et al., 1989, Molecular cloning: A laboratory manual, 2nd edition, Cold Spring Harbor Laboratory Press, NY, USA), its specificity or the properties of the transport mechanism are changed. The strain MG887ura3 ^~ :: pFL61-PUP1 is directly suitable for the investigation of inhibitors or promoters of nucleobase transport.

The PUP1 protein expressed by the yeast strain M887ura3 ^~ :: pFL61-PUP1 was subjected to a hydrophobicity analysis according to Kyte and Doolittle. As can be seen from FIG. 1, the PUP1 protein 9-12 has strongly hydrophobic regions (positive values on the Y axis) which are long enough to cross the membrane once. The first hydrophobic region is not conserved in all PUP proteins.

Sequence analysis of the cDNA insertion of the plasmid PFL61-PUP1

The plasmid pFL61-PUP1 was isolated from the yeast strain MG887ura3 ^~ :: pFL61 -PUP1 and the insertion was carried out using synthetic oligonucleotides according to the method of Sanger et al. (1977, Proc. Natl. Acad. Sci. USA 74, 5463-5467). The coding sequence of the PUP1 gene is reproduced in SEQ ID NO 1 and the protein sequence derived therefrom in SEQ ID NO 8. Example 2: Recording studies with ¹⁴ C-labeled adenine, ¹⁴ C-labeled cytosine and ³ H-labeled cvtokinins on the yeast strain MG887ura3 ^~ :: pFL61-PUP1

To measure the uptake rates, the yeast strains MG887ura3 ^~ :: pFL61, MG887ura3 ^~ :: pFL61-PUP1 and their parent strain ∑1278b (Dubois & Grenson, 1979, Mol. Gen. Genet. 175, 67-76) were used in full medium ( YPD) without uracil and with 2% glucose as carbon source up to an OD ₆ oo of 0.6 at 28 ° C. The cells were harvested at 3000 g, washed twice with water and adjusted to an OD600 of 12 with sodium phosphate buffer (100 mM, pH 4.5), 1% glucose. The cell suspension was stored in 100 μl portions on ice until the start of the uptake experiment. Before the start of the uptake measurements, the cells were preincubated at 30 ° C. for 2 min. The reaction was then started by adding 100 μl of the radioactively labeled substrate solution to 100 μl of cell suspension. The reaction mixture was incubated at 30 ° C., and after 30, 60, 120, 180 seconds, 50 μl of the suspension was removed in each case and added to 4 ml of ice-cold water (for measurements over 180 seconds, more cell suspension and substrate solution were used accordingly). The cells were sucked onto glass fiber filters and washed with 4 ml of ice-cold water. The radioactivity on the filters was then determined in the liquid scintillation counter.

Substrate solution: The substrate solution is a 100 mM sodium phosphate buffer pH 4.5 with 1% glucose and 9.25 Bq / μl of the radiolabelled substrate (25-100 μM [ ¹ C] adenine or [ ¹⁴ C] cytosine ). In addition, the substrate solution contains the unlabelled substrate, any inhibitors and competitors in a double concentration. To determine the optimum pH, the pH of the sodium phosphate buffer was also changed. When measuring the influence of glucose on the uptake rates, neither the cell suspension nor the substrate solution contained glucose. Glucose was only added to the measurement batch in a final concentration of 1% after the start of the uptake measurements. Furthermore it could be shown that PUP2 is also able to transport adenine.

Table 1 shows the uptake of radioactively labeled adenine and cytosine by the yeast strain MG887ura3 ~ :: pFL61- mediated by the PUP1 nucleobase transporter. PUP1 specified. To calculate the intracellular concentration, the cell volume was estimated to be four times the dry weight (Ninnemann et al, 1994, EMBO J15, 3464-3471). The recording takes place against a concentration gradient. ^~~

Table 1

Initial conc. Final conc. Final conc. Enrichment Medium Medium Cells Factor

Adenine 200 uM 155 uM 2350 uM 15 Cytosine 200 uM 145 uM 2660 uM 18

The influence of various inhibitors on the adenine or cytosine uptake mediated by the PUP1 nucleobase transporter in the yeast strain MG887ura3 ^~ :: pFL61-PUP1 is shown in Table 2. The inhibitors were added to the cells five minutes before the start of the measurements. The protonophores carbonyl-cyanide-m-chlorophenylhydratzon (CCCP) and 2,4-dinitrophenol (2,4-DNP) and the HVATPase inhibitor diethylstilbestrol block the uptake. This can be seen as a clear indication of a secondary active, proton-coupled uptake mechanism.

Table 2

FIG. 2 shows that the cytosine uptake into the yeast strain MG877ura3 ~ :: pFL61-PUP1 mediated by the PUP1 nucleobase transporter is dependent on the pH value. Similar results were obtained for adenine uptake (data not shown). As shown in Figure 3, the cytosine uptake mediated by the PUP1 nucleobase transporter in the yeast strain MG877ura3 ^~ :: pFL61-PUP1 is glucose dependent. Similar results were obtained for adenine uptake (data not shown). The increase in activity when glucose is added can be used as an indication of energy dependence. This, as well as the observed increase in activity with decreasing pH value, indicates a secondary active, proton-coupled uptake mechanism.

To investigate the substrate specificity of the PUP1 nucleobase transporter expressed in yeast in comparison to the yeast's own FCY2 transporter, competition experiments were carried out with non-radioactive Subtrateπ. The results of these experiments are shown in Fig. 4. The uptake of radioactively labeled adenine was measured and set as 100% (corresponds to 1, 7 or 0.9 nmol.minNmg ^"1 dry weight). The measurements were carried out at a substrate concentration of 25 μM. The competitors were used in a 10-fold molar excess .

To analyze the competitive inhibition of the adenine transport mediated by the PUP1 nucleobase transporter by the cytokinins kinetin and zeatin, uptake experiments were carried out with different concentrations of radioactively labeled adenine and different competitor concentrations. The inhibitor constants Ki were determined from Lineweaver / Burk plots (35 μM for kinetin and 30 μM for zeatin).

Direct uptake experiments in PUP1-expressing yeast cells with ³ H-labeled cytokinins (zeatin and 2-isopentenyladenine) show that PUP1 can also transport cytokinins (FIG. 5). The uptake experiments were carried out with ³ H-labeled zeatin in MG887ura3 ^~ as for adenine. 87.3 Bq / μl of the radioactive substrate were used per reaction mixture at a total concentration of 100 μM trans-zeatin. The results of three independent experiments showed that the PUP1-expressingπ yeast clones compared to the control strains transformed with the control plasmid pDR195 showed a 70-fold increase in radioactively labeled trans-zeatin. This shows that PUP1 mediates the transport of trans-zeatin. Example 3: Transformation of plants with constructions for overexpression or antisense repression of a nucleobase transporter gene

The introduction of a nucleic acid coding for a plant nucleobase transporter and the overexpression or antisense repression of a nucleobase transporter gene for changing the transport of nucleobases and their derivatives is described here using the example of Arabidopsis thaliana. The application is not limited to this species.

a) Overexpression: The 1.2 kb Λ / oW fragment from the plasmid pFL61-PUP1, which contains the cDNA as an insert for a nucleobase transporter from Arabidopsis thaliana, was cloned into the vector pT7T3 18U / Λ / of / cut with NotI. The vector ρT7T3 18U / Λ / 0 «was generated by inserting a Notl-Unker into the St77a / interface of pT7T3 18U (Pharmacia). The orientation of the fragment was checked by restriction cleavage. The vector contains a Kpnl and an Xöa / interface in the multiple cloning site so that the PUP1 cDNA could be isolated from this vector as a pn / -Xi-> a / fragment. This 1.2kb Kpnl-Xbal fragment was cloned into the Kpnl-Xbal cut vector pBinAR, a derivative of pBIN19 (Bevan, 1984, Nucl. Acids Res. 12, 8711-8720). The resulting plasmid was named p35S-PUP1.

b) Antisense repression: A 1.1 kb Hpal-Ssp / -PUP1 fragment was isolated from the plasmid pFL61-PUP1 and cloned into the Sma / interface of pBinAR in an antisense orientation. The orientation of the fragment was checked by restriction cleavage. The resulting plasmid was named p35-α-PUP1.

The PUP1 cDNA fragments have the designation "B" in FIGS. 6a and 6b. Depending on whether B was introduced in the sense of the CaMV-35S promoter from pBinAR or not, the resulting plasmid is called p35S-PUP1 or p35S-α-PUP1. A fragment from the cauliflower mosaic virus genome carrying the 35S promoter (nt 6909-7437) was inserted between its EcoRI and pt7 / interfaces. The promoter fragment was prepared as an EcoR // pn / fragment from the plasmid pDH51 (Pietrzak et al., Nucl. Acids Res. 14, 5857-5868). In the plasmid card, the promoter fragment is labeled "A". Between the sink and the / - // t7α7 // - interface of pBinAR the polyadenylation signal of gene 3 of the T-DNA of the plasmid pTiACHδ (Gielen et al., EMBO J. 3, 835-846) is also inserted. For this purpose, a Pvull / Hindlll fragment (nt 11749-11939) from the plasmid pAGV 40 (Herrera-Estrella et al., 1983, Nature 303, 209-2139) was at the Pvu // interface with a Sp / 7 / - Left has been provided. The polyadenylation signal is labeled "C" in the plasmid card.

After transformation of agrobacteria with the plasmids p35S-PUP1 and p35S-α-PUP1, these were used for vacuum infiltration of Arabidopsis thaliana.

Ten independently obtained transformants for both constructs, in which the presence of the intact, non-rearranged chimeric gene had been proven by means of "Southern blot" analyzes, were examined for the changes in the nucleobase transport.

Example 4: Expression of the PUP1 gene in Xenoptys oocytes

The cDNA for PUP1 was cut with NotI and cloned into an oocyte expression vector which contains untranslated 5 'and 3' regions of the β globi from Xenopus. The cDNA was then amplified using PCR. SP6 (from the oocyte vector) was used as the forward primer, and a primer which destroys the STOP codon of PUP1 and at the same time inserts a / Votf interface was used as the backward primer. This PCR fragment was cut into Notl and Hindill and ligated into an oocyte expression vector which additionally contains the gene for GFP ("green fiourescent protein" from the jellyfish) behind the Notl interface. This results in an open reading for a fusion protein from PUP1 and GFP The linker with three base pairs coding for alanine is formed by inserting the / Votf interface, the plasmid has been linearized with Mlul, and RNA has been transcribed in vitro using SP6 polymerase and taken up to about 1 ng / nl in water per oocyte (maturity stage 5 to 6) 50 nl were injected in. After two days of incubation at 16 °, oocytes were placed in a corresponding measuring chamber for measurement and rinsed with various solutions, which contained: 100 mM N-methyl-D-glutamine chloride, 2 mM calcium chloride, 5 mM MES, pH 5.0 or 7.3 and corresponding concentrations of adenine. A glass electrode (filled with 3 M KCI) was inserted into the oocyte for voltage measurement. The potential was measured relative to the reference electrode using a Dagan amplifier. This was connected to the bath solution via an agar bridge. In solutions without adenine at pH 7.3, oocytes had a typical resting potential of approximately - 30 mV (see FIG. 7). Adenine in the bath solution causes depolarization of the oocyte. This shows that PUP1 also transports adenine in oocytes.

The oocyte was clamped to a voltage of -80 mV for current measurement. Adenine in the bath solution causes an inward flow. Since adenine itself is not charged, this indicates cotransport with charged ions, e.g. Protons,

Example 5: PUP1 -mediated transport of adenosine in yeast

The Saccharomyces cerev / s / ^' ae strain DM734-284D (genotype: ade8-18, ade2-1. Arg4-16, leu2-27, trp1-1, Iys2, ura3 Yeast Geπetic Stock Center) was used for growth studies. Due to the mutations in the gene loci ade 8-18 and ade 2-1, this yeast strain is unable to synthesize adenine itself. This strain therefore needs adenine from the external medium to be able to grow. This adenine is taken up via the yeast purine / cytosine transporter FCY2. However, the strain DM734-284D is also able to grow on medium which contains adenosine instead of adenine, provided that adenosine transport into the yeast cell is mediated by a genetically engineered adenosine transporter. Saccharomyces cerevisiae itself does not have such an adenosine transporter. The strain DM734-284D is therefore suitable as a complementation system for isolating adenosine transporters from various organisms.

To check whether PUP1 mediates the transport of adenosine, the strain DM734-284D was transformed with the constructs pDR195PUP1 and pDR195 as a control (Rentsch et al., 1995, FEBS Lett. 370, 264-368) according to the standard protocol for yeast transformation (Dohmen et al., 1991, Yeast 7, 691-692). The transformation approaches were then plated on minimal medium to check the transformation, which contained 150 μM adenine and the amino acids arginine, leucine, tryptophan (each 60 mg / l) and lysine (70 mg / l) required for growth. Uracil served as the transformation marker. Three independent positive clones of the control and the PUP1-expressing clones were plated on minimal medium which contained 150 μM adenosine instead of 150 μM adenine. The clones expressing PUP1-1 showed significant growth after three days, while the clones transformed with the vector pDR 195 showed no growth (see FIG. 8). This shows that PUP1 mediates the transport of the nucleoside and adenine derivative adenosine.

Example 6: PUP1 -mediated transport of caffeine in yeast

In order to check whether PUP1 mediates the transport of caffeine, the sensitivity of the Saccharomyces cerev / s / ae strain MG877ura3 ^~ on caffeine-containing minimal medium was determined. From certain concentrations, caffeine has a toxic effect on yeast (Bard et al., 1980, J. Bacteriol. 141, 999-1002), which leads to slower growth or death of the yeast. If PUP1 mediates the transport of caffeine, it can be assumed that a yeast strain which expresses this protein is more sensitive to caffeine than the corresponding control strain, which manifests itself in reduced growth.

Various caffeine concentrations between 0 to 1.5% caffeine in minimal medium were used in this test. Yeast clones grown in liquid minimal medium for 16 hours were streaked on the appropriate plates and incubated for 6 days at 28 ° C. It was found that yeast expressing PUP1 showed a significantly slower growth compared to the control strain MG877ura3 ^~ and the PUP2 expressing strain from the concentration of 0.2% caffeine (see FIG. 9). This must be attributed to the increased intake of toxic caffeine and shows that PUP1 mediates the transport of caffeine.

Claims

claims

1. Nucleic acid encoding a plant or animal nucleobase transporter selected from:

a) nucleic acid which is obtainable by complementing nucleobase transporter-deficient host cells with a plant or animal gene bank and selection for nucleobase transporter-positive host cells;

b) nucleic acid with a sequence which codes for a protein with a sequence according to SEQ ID NO 8 or SEQ ID NO 9;

c) nucleic acid which hybridizes with a nucleic acid according to b);

d) nucleic acid which, taking into account the degeneration of the genetic code, would hybridize with a nucleic acid according to b) or with the sequence complementary to b);

e) derivatives of a nucleic acid according to a) to d) obtained by substitution, addition, inversion and / or deletion of one or more bases;

except for nucleic acids with a sequence according to one of SEQ ID NO 3 to 5.

2. Nucleic acid according to claim 1, characterized in that it comprises the coding sequence of one of the sequences according to SEQ ID NO 1, 2, 6, 7 or 10 or one of these by substitution, addition, inversion and / or deletion of one or more Base derived derivative.

3. Nucleic acid according to one of claims 1 or 2, characterized in that it is a DNA.

4. Fragment of a nucleic acid according to one of claims 1 to 3, characterized in that it can inhibit the expression of a nucleobase transporter in a host cell in anti-orientation to a promoter.

5. Fragment according to claim 4, characterized in that it comprises at least 10 nucleotides, preferably at least 50 nucleotides, particularly preferably at least 200 nucleotides.

6. Construct containing a nucleic acid according to one of claims 1 to 3 and / or a fragment according to one of claims 4 or 5 under the control of the expression regulating elements.

7. Construct according to claim 6, characterized in that the nucleic acid or the fragment is in the anti-sense orientation to the regulatory element.

8. Construct according to one of claims 6 or 7, characterized in that it is present in a plasmid.

9. host cell containing a nucleic acid according to one of claims 1 to 3 and / or a nucleic acid with a sequence according to one of SEQ ID NO 3 to 5 and / or a fragment of the aforementioned nucleic acids and / or a construct according to one of claims 6 to 8 .

10. Host cell according to claim 9, characterized in that it is selected from bacteria, yeast cells, mammalian cells and plant cells.

11. Transgenic plant and parts of plants and / or seeds of the plant containing a nucleic acid according to one of claims 1 to 3 and / or a nucleic acid with a sequence according to one of SEQ ID NO 3 to 5 and / or a fragment of the aforementioned nucleic acids and / or a construct according to any one of claims 6 to 8.

12. Transgenic plant, plant part, host cell and / or seed according to one of claims 9 to 11, characterized in that the nucleic acid or the fragment or the construct is integrated into the genome at a location that does not correspond to its natural position.

13. Protein obtainable by expression of a nucleic acid according to one of claims 1 to 3 or a nucleic acid with a sequence according to one of SEQ ID NO 3 to 5 in a host cell.

14. Antibody that reacts with a protein according to claim 13.

15. A method for producing a transgenic plant, comprising the following steps:

Introducing a nucleic acid according to one of claims 1 to 3 or a nucleic acid with a sequence according to one of SEQ ID NO 3 to 5 and / or a fragment of these nucleic acids into a plant cell; and

- Regeneration of a plant from the transformed plant cell.

16. A method for influencing the nucleobase transporter properties of a plant, a plant part, a plant cell and / or seeds, which comprises the step:

- Introducing a nucleic acid according to one of claims 1 to 3 or a nucleic acid with a sequence according to one of SEQ ID NO 3 to 5 and / or a fragment of these nucleic acids in a plant cell and / or a plant.

17. Use of a plant cell according to claim 10 for the regeneration and production of whole plants.

18. Use of a nucleic acid according to one of claims 1 to 3 or a nucleic acid with a sequence according to one of SEQ ID NO 3 to 5 for the isolation of homologous sequences from bacteria, fungi, plants, animals and / or humans.

19. Use of a nucleic acid according to one of claims 1 to 3 or a nucleic acid with a sequence according to one of SEQ ID NO 3 to 5 for the expression of a nucleobase transporter in pro- and / or eukaryotic cells.

20. Use of a nucleic acid according to one of claims 1 to 3 or a nucleic acid with a sequence according to one of SEQ ID NO 3 to 5 under the control of a regulatory element in antisense orientation to inhibit the expression of an endogenous nucleobase transporter in pro- and / or eukaryotic cells .

21. Use of a nucleic acid according to one of claims 1 to 3 or a nucleic acid with a sequence according to one of SEQ ID NO 3 to 5 for the production of transgenic useful plants.

22. Use of a nucleic acid according to one of claims 1 to 3 or a nucleic acid with a sequence according to one of SEQ ID NO 3 to 5 for the identification of inhibitors of nucleobase transport.