Abstract:
There are described DNA sequences that contain the coding region of amino acid transporters whose introduction in a plant genome modifies the transfer of metabolites in transgenic plants, plasmids, bacteria, yeasts and plants containing these DNA sequences, as well as their use.

Description:
BACKGROUND OF THE INVENTION  
       FILED OF THE INVENTION  
         [0001]    The present invention relates to DNA sequences that contain the coding region of amino acid transporters, whose introduction in a plant genome modifies the transfer of metabolites in transgenic plants, plasmids, bacteria, yeasts and plants containing these DNA sequences, as well as their use.  
           [0002]    For many plant species it is known that the delivery of energy-rich compounds to the phloem through the cell wall takes place throughout the cell. Transporter molecules which allow the penetration of amino acids through the plant cell wall are not known.  
           [0003]    In bacteria, numerous amino acid transport systems have been characterized. For aromatic amino acids, 5 different transporters have been described which can transport any one of phenylalanine, tyrosine and tryptophan, while the other transporters are specific for individual amino acids (see Sarsero et al., 1991, J Bacteriol 173: 3231-3234). The speed constants of the transport process indicates that the specific transport is less efficient. For several transporter proteins, the corresponding genes have been cloned. This has been achieved using transport-deficient mutants which were selected for their transport ability after transformation with DNA fragments as inserts in expression vectors (see Wallace et al., 1990, J Bacteriol 172: 3214-3220). The mutants were selected depending on their ability to grow in the presence of toxic analogues of amino acids, since the mutants cannot take these up and therefore cannot be impaired.  
           [0004]    Corresponding complementation studies have been carried out with the eukaryotic yeast,  Saccharomyces cerevisiae . Tanaka &amp; Fink (1985, Gene 38: 205-214) describe a histidine transporter that was identified by complementation of a mutation. Vandenbol et al. (1989, Gene 83: 153-159) describe a proline transporter for  Saccharomyces cerevisiae.  The yeast possesses two different permeases for proline. One transports with lower efficiency and can be used also for other amino acids, and the other is proline-specific and works with high affinity. The latter was coded from the put4 gene. This carries an open reading frame for a peptide with a molecular weight of 69 kDa. The protein contains 12 membrane-penetrating regions, but does not contain any N-terminal signal sequence for secretion. This is a typical property of integral membrane proteins. The permeases process homology for arginine and for histidine permease from yeast, but not, however, for proline permease from  Escherichia coli.    
           [0005]    For plant cells, based on studies on tobacco suspension cultures, it has been found that the transport of arginine, asparagine, phenylalanine and histidine are pH and energy dependent. Since a 1,000-fold excess of leucine inhibits the transport of the other amino acids, it can be assumed, therefore, that all amino acids use the same transporter (McDaniel et al., 1982, Plant Physio 69: 246-249). Li and Bush (1991, Plant Physiol 96: 1338-1344) determined, for aliphatic, neutral amino acids, two transport systems in plasma membrane vesicles from  Beta vulgaris . On the one hand, alanine, methionine, glutamine and leucine displace each other on the transporter protein. On the other hand, isoleucine, valine and threonine have mutually competitive effects. In combined competition kinetic studies (Li &amp; Bush, 1990, Plant Physiol 94: 268-277) four different transport systems have been distinguished. Besides a transporter for all neutral amino acids, which work with low affinity, there exists a high affinity type which, however, possesses low affinity for isoleucine, threonine, valine and proline. Further transporters exist for acids as well as for basic amino acids.  
           [0006]    The transporter molecule or gene for plant transporter proteins is not known.  
         SUMMARY OF THE INVENTION  
         [0007]    There are now described DNA sequences which contain the coding region of a plant amino acid transporter, and whose information contained in the nucleotide sequence allows, by integration in a plant genome, the formation of RNA, by which a new amino acid transport activity can be introduced in the plant cells or an endogenous amino acid transporter activity can be expressed.  
           [0008]    Under the term amino transporter is to be understood, for example a cDNA sequence that codes an amino transporter from  Arabidopsis thaliana.    
           [0009]    The identification of the coding region of the amino acid transporter is carried out by a process which allows the isolation of plant DNA sequences which code transporter molecules by means of expression in specific mutants of yeast  Saccharomyces cerevisiae.  For this, suitable yeast mutants have to be provided which cannot take up a substance for which the coding region of the transporter molecule has to be isolated from a plant gene library.  
           [0010]    A mutant which cannot grow in media, with proline or citrulline as the only nitrogen source, is described by Jauniaux et al. (1987), Eur J Biochem 164: 601-606).  
           [0011]    For the preparation of yeast strains that can be used to identify plant amino acid transporters, a yeast mutant which is not able to grow in media with proline and/or citrulline as the only nitrogen source is, for example, transformed with pFL 61 plasmid, which carries, as an insert, cDNA fragments from a cDNA library from  Arabidopsis thaliana.    
           [0012]    Further, a double mutant JT16 (Tanaka &amp; Fink, 1985, Gene 38: 205-214) which has a deficiency in histidine synthesis (his4) and in histidine.uptake (hip1) is transformed with the described pFL 61 plasmid and cultivated in a medium with addition of histidine.  
           [0013]    It has now surprisingly been found that, in the transformation of yeast cells, certain plant cDNA fragments can complement the yeast mutation. By analysis of the properties of the proteins coded from the cDNA it can be shown that a coding region that codes a plant amino acid transporter with a wide specificity spectrum is responsible for the complementing of the mutation (see example 3).  
           [0014]    Such a coding region of an amino acid transporter is shown, for example, by one of the following nucleotide sequences:  
                                           1. Sequence:                   CTTAAAACAT TTATTTTATC TTCTTCTTGT TCTCTCTTTC TCTTTCTCTC ATCACT   56   (Seq. ID No. 1)               ATG AAG AGT TTC AAC ACA GAA GGA CAC AAC CAC TCC ACG GCG GAA   101               Met Lys Ser Phe Asn Thr Glu Gly His Asn His Ser Thr Ala Glu       1               5                   10                  15               TCC GGC GAT GCC TAC ACC GTG TCG GAC CCG ACA AAG AAC GTC GAT   146               Ser Gly Asp Ala Tyr Thr Val Ser Asp Pro Thr Lys Asn Val Asp                       20                  25                  30               GAA GAT GGT CGA GAG AAG CGT ACC GGG ACG TGG CTT ACG GCG AGT   191               Glu Asp Gly Arg Glu Lys Arg Thr Gly Thr Trp Leu Thr Ala Ser                       35                  40                  45               GCG CAT ATT ATC ACG GCG GTG ATA GGC TCC GGA GTG TTG TCT TTA   236               Ala His Ile Ile Thr Ala Val Ile Gly Ser Gly Val Leu Ser Leu                       50                  55                  60               GCA TGG GCT ATA GCT CAG CTT GGT TGG ATC GCA GGG ACA TCG ATC   281               Ala Trp Ala Ile Ala Gln Leu Gly Trp Ile Ala Gly Thr Ser Ile                       65                  70                  75               TTA CTC ATT TTC TCG TTC ATT ACT TAC TTC ACC TCC ACC ATG CTT   326               Leu Leu Ile Phe Ser Phe Ile Thr Tyr Phe Thr Ser Thr Met Leu                       80                  85                  90               GCC GAT TGC TAC CGT GCG CCG GAT CCC GTC ACC GGA AAA CGG AAT   371               Ala Asp Cys Tyr Arg Ala Pro Asp Pro Val Thr Gly Lys Arg Asn                       95                  100                 105               TAC ACT TAC ATG GAC GTT GTT CGA TCT TAC CTC GGT GGT AGG AAA   416               Tyr Thr Tyr Met Asp Val Val Arg Ser Tyr Leu Gly Gly Arg Lys                      110                 115                 120               GTG CAG CTC TGT GGA GTG GCA CAA TAT GGG AAT CTG ATT GGG GTC   461               Val Gln Leu Cys Gly Val Ala Gln Tyr Gly Asn Leu Ile Gly Val                       125                 130                 135               ACT GTT GGT TAC ACC ATC ACT GCT TCT ATT AGT TTG GTA GCG GTA   506               Thr Val Gly Tyr Thr Ile Thr Ala Ser Ile Ser Leu Val Ala Val                       140                 145                 150               GGG AAA TCG AAC TGC TTC CAC GAT AAA GGG CAC ACT GCG GAT TGT   551               Gly Lys Ser Asn Cys Phe His Asp Lys Gly His Thr Ala Asp Cys                       155                 160                 165               ACT ATA TCG AAT TAT CCG TAT ATG GCG GTT TTT GGT ATC ATT CAA   596               Thr Ile Ser Asn Tyr Pro Tyr Met Ala Val Phe Gly Ile Ile Gln                       170                 175                 180               GTT ATT CTT AGC CAG ATC CCA AAT TTC CAC AAG CTC TCT TTT CTT   641               Val Ile Leu Ser Gln Ile Pro Asn Phe His Lys Leu Ser Phe Leu                       185                 190                 195               TCC ATT ATG GCC GCA GTC ATG TCC TTT ACT TAT GCA ACT ATT GGA   686               Ser Ile Met Ala Ala Val Met Ser Phe Thr Tyr Ala Thr Ile Gly                       200                 205                 210               ATC GGT CTA GCC ATC GCA ACC GTC GCA GGT GGG AAA GTG GGT AAG   731               Ile Gly Leu Ala Ile Ala Thr Val Ala Gly Gly Lys Val Gly Lys                       215                 220                 225               ACG AGT ATG ACG GGC ACA GCG GTT GGA GTA GAT GTA ACC GCA GCT   776               Thr Ser Met Thr Gly Thr Ala Val Gly Val Asp Val Thr Ala Ala                       230                 235                 240               CAA AAG ATA TGG AGA TCG TTT CAA GCG GTT GGG GAC ATA GCG TTC   821               Gln Lys Ile Trp Arg Ser Phe Gln Ala Val Gly Asp Ile Ala Phe                       245                 250                 255               GCC TAT GCT TAT GCC ACG GTT CTC ATC GAG ATT CAG GAT ACA CTA   866               Ala Tyr Ala Tyr Ala Thr Val Leu Ile Glu Ile Gln Asp Thr Leu                       260                 265                 270               AGA TCT AGC CCA GCT GAG AAC AAA GCC ATG AAA AGA GCA AGT CTT   911               Arg Ser Ser Pro Ala Glu Asn Lys Ala Met Lys Arg Ala Ser Leu                       275                 280                 285               GTG GGA GTA TCA ACC ACC ACT TTT TTC TAC ATC TTA TGT GGA TGC   956               Val Gly Val Ser Thr Thr Thr Phe Phe Tyr Ile Leu Cys Gly Cys                       290                 295                 300               ATC GGC TAT GCT GCA TTT GGA AAC AAT GCC CCT GGA GAT TTC CTC   1001               Ile Gly Tyr Ala Ala Phe Gly Asn Asn Ala Pro Gly Asp Phe Leu                       305                 310                 315               ACA GAT TTC GGG TTT TTC GAG CCC TTT TGG CTC ATT GAC TTT GCA   1046               Thr Asp Phe Gly Phe Phe Glu Pro Phe Trp Leu Ile Asp Phe Ala                       320                 325                 330               AAC GCT TGC ATC GCT GTC CAC CTT ATT GGT GCC TAT CAG GTG TTC   1091               Asn Ala Cys Ile Ala Val His Leu Ile Gly Ala Tyr Gln Val Phe                       335                 340                 345               GCG CAG CCG ATA TTC CAG TTT GTT GAG AAA AAA TGC AAC AGA AAC   1136               Ala Gln Pro Ile Phe Gln Phe Val Glu Lys Lys Cys Asn Arg Asn                       350                 355                 360               TAT CCA GAC AAC AAG TTC ATC ACT TCT GAA TAT TCA GTA AAC GTA   1181               Tyr Pro Asp Asn Lys Phe Ile Thr Ser Glu Tyr Ser Val Asn Val                       365                 370                 375               CCT TTC CTT GGA AAA TTC AAC ATT AGC CTC TTC AGA TTG GTG TGG   1226               Pro Phe Leu Gly Lys Phe Asn Ile Ser Leu Phe Arg Leu Val Trp                       380                 385                 390               AGG ACA GCT TAT GTG GTT ATA ACC ACT GTT GTA GCT ATG ATA TTC   1271               Arg Thr Ala Tyr Val Val Ile Thr Thr Val Val Ala Met Ile Phe                       395                 400                 405               CCT TTC TTC AAC GCG ATC TTA GGT CTT ATC GGA GCA GCT TCC TTC   1316               Pro Phe Phe Asn Ala Ile Leu Gly Leu Ile Gly Ala Ala Ser Phe                       410                 415                 420               TGG CCT TTA ACG GTT TAT TTC CCT GTG GAG ATG CAC ATT GCA CAA   1361               Trp Pro Leu Thr Val Tyr Phe Pro Val Glu Met His Ile Ala Gln                       425                 430                 435               ACC AAG ATT AAG AAG TAC TCT GCT AGA TGG ATT GCG CTG AAA ACG   1406               Thr Lys Ile Lys Lys Tyr Ser Ala Arg Trp Ile Ala Leu Lys Thr                       440                 445                 450               ATG TGC TAT GTT TGC TTG ATC GTC TCG CTC TTA GCT GCA GCC GGA   1451               Met Cys Tyr Val Cys Leu Ile Val Ser Leu Leu Ala Ala Ala Gly                       455                 460                 465               TCC ATC GCA GGA CTT ATA AGT AGT GTC AAA ACC TAC AAG CCC TTC   1496               Ser Ile Ala Gly Leu Ile Ser Ser Val Lys Thr Tyr Lys Pro Phe                       470                 475                 480               CGG ACT ATG CAT GAG TGAGTTTGAG ATCCTCAAGA GAGTCAAAAA   1541               Arg Thr Met His Glu                       485               TATATGTAGT AGTTTGGTCT TTCTGTTAAA CTATCTGGTG TCTAAATCCA   1591               ATGAGAATGC TTTATTGCTA AAACTTCATG AATCTCTCTG TATCTACATC   1641               TTTCAATCTA ATACATATGA GCTCTTCCAA AAAAAAAAAA AAAA   1685          
 
           [0015]    [0015]                                           2. Sequence:                                         CTATTTTAT AATTCCTCTT CTTTTTTGTTC   29   (Seq. ID No. 2)                ATAGCTTTGT AATTATAGTC TTATTTCTCT TTAAGGCTCA ATAAGAGGAG   79               ATG GGT GAA ACC GCT GCC GCC AAT AAC CAC CGT CAC CAC CAC CAT   124               Met Gly Glu Thr Ala Ala Ala Asn Asn His Arg His His His His       1               5                   10                  15               CAC GGC CAC CAG GTC TTT GAC GTG GCC AGC CAC GAT TTC GTC CCT   169               His Gly His Gln Val Phe Asp Val Ala Ser His Asp Phe Val Pro                       20                  25                  30               CCA CAA CCG GCT TTT AAA TGC TTC GAT GAT GAT GGC CCC CTC AAA   214               Pro Gln Pro Ala Phe Lys Cys Phe Asp Asp Asp Gly Arg Leu Lys                       35                  40                  45               AGA ACT GGG ACT GTT TGG ACC GCG AGC GCT CAT ATA ATA ACT GCG   259               Arg Thr Gly Thr Val Trp Thr Ala Ser Ala His Ile Ile Thr Ala                       50                  55                  60               GTT ATC GGA TCC GGC GTT TTG TCA TTG GCG TGG GCG ATT GCA CAG   304               Val Ile Gly Ser Gly Val Leu Ser Leu Ala Trp Ala Ile Ala Gln                       65                  70                  75               CTC GGA TGG ATC GCT GGC CCT GCT GTG ATG CTA TTG TTC TCT CTT   349               Leu Gly Trp Ile Ala Gly Pro Ala Val Met Leu Leu Phe Ser Leu                       80                  85                  90               GTT ACT CTT TAC TCC TCC ACA CTT CTT AGC GAC TGC TAC AGA ACC   394               Val Thr Leu Tyr Ser Ser Thr Leu Leu Ser Asp Cys Tyr Arg Thr                       95                  100                 105               GGC GAT GCA GTG TCT GGC AAC AGA AAC TAC ACT TAC ATG GAT GCC   439               Gly Asp Ala Val Ser Gly Lys Arg Asn Tyr Thr Tyr Met Asp Ala                       110                 115                 120               GTT CGA TCA ATT CTC GGT GGG TTC AAG TTC AAG ATT TGT GGG TTG   484               Val Arg Ser Ile Leu Gly Gly Phe Lys Phe Lys Ile Cys Gly Leu                       125                 130                 135               ATT CAA TAC TTG AAT CTC TTT GGT ATC GCA ATT GGA TAC ACG ATA   529               Ile Gln Tyr Leu Asn Leu Phe Gly Ile Ala Ile Gly Tyr Thr Ile                       140                 145                 150               GCA GCT TCC ATA AGC ATG ATG GCG ATC AAG AGA TCC AAC TGC TTC   574               Ala Ala Ser Ile Ser Met Met Ala Ile Lys Arg Ser Asn Cys Phe                       155                 160                 165               CAC AAG AGT GGA GGA AAA GAC CCA TGT CAC ATG TCC AGT AAT CCT   619               His Lys Ser Gly Gly Lys Asp Pro Cys His Met Ser Ser Asn Pro                       170                 175                 180               TAC ATG ATC GTA TTT GGT GTG GCA GAG ATC TTG CTC TCT CAG GTT   664               Tyr Met Ile Val Phe Gly Val Ala Glu Ile Leu Leu Ser Gln Val                       185                 190                 195               CCT GAT TTC GAT CAG ATT TGG TGG ATC TCC ATT GTT GCA GCT GTT   709               Pro Asp Phe Asp Gln Ile Trp Trp Ile Ser Ile Val Ala Ala Val                       200                 205                 210               ATG TCC TTC ACT TAC TCT GCC ATT GGT CTA GCT CTT GGA ATC GTT   754               Met Ser Phe Thr Tyr Ser Ala Ile Gly Leu Ala Leu Gly Ile Val                       215                 220                 225               CAA GTT GCA GCG AAT GGA GTT TTC AAA GGA AGT CTC ACT GGA ATA   799               Gln Val Ala Ala Asn Gly Val Phe Lys Gly Ser Leu Thr Gly Ile                       230                 235                 240               AGC ATC GGA ACA GTG ACT CAA ACA CAG AAG ATA TGG AGA ACC TTC   844               Ser Ile Gly Thr Val Thr Gln Thr Gln Lys Ile Trp Arg Thr Phe                       245                 250                 255               CAA GCA CTT GGA GAC ATT GCC TTT GCG TAC TCA TAC TCT GTT GTC   889               Gln Ala Leu Gly Asp Ile Ala Phe Ala Tyr Ser Tyr Ser Val Val                       260                 265                 270               CTA ATC GAG ATT CAG GAT ACT GTA AGA TCC CCA CCG GCG GAA TCG   934               Leu Ile Glu Ile Gln Asp Thr Val Arg Ser Pro Pro Ala Glu Ser                       275                 280                 285               AAA ACG ATG AAG AAA GCA ACA AAA ATC AGT ATT GCC GTC ACA ACT   979               Lys Thr Met Lys Lys Ala Thr Lys Ile Ser Ile Ala Val Thr Thr                       290                 295                 300               ATC TTC TAC ATG CTA TGT GGC TCA ATG GGT TAT GCC GCT TTT GGA   1024               Ile Phe Tyr Met Leu Cys Gly Ser Met Gly Tyr Ala Ala Phe Gly                       305                 310                 315               GAT GCA GCA CCG GGA AAC CTC CTC ACC GGT TTT GGA TTC TAC AAC   1069               Asp Ala Ala Pro Gly Asn Leu Leu Thr Gly Phe Gly Phe Tyr Asn                       320                 325                 330               CCG TTT TGG CTC CTT GAC ATA GCT AAC GCC GCC ATT GTT GTC CAC   1114               Pro Phe Trp Leu Leu Asp Ile Ala Asn Ala Ala Ile Val Val His                       335                 340                 345               CTC GTT GGA GCT TAC CAA GTC TTT GCT CAG 000 ATC TTT GCC TTT   1159               Leu Val Gly Ala Tyr Gln Val Phe Ala Gln Pro Ile Phe Ala Phe                       350                 355                 360               ATT GAA AAA TCA GTC GCA GAG AGA TAT CCA GAC AAT GAC TTC CTC   1204               Ile Glu Lys Ser Val Ala Glu Arg Tyr Pro Asp Asn Asp Phe Leu                       365                 370                 375               AGC AAG GAA TTT GAA ATC AGA ATC CCC GGA TTT AAG TCT CCT TAC   1249               Ser Lys Glu Phe Glu Ile Arg Ile Pro Gly Phe Lys Ser Pro Tyr                       380                 385                 390               AAA GTA AAC GTT TTC AGG ATG GTT TAC AGG AGT GGC TTT GTC GTT   1294               Lys Val Asn Val Phe Arg Met Val Tyr Arg Ser Gly Phe Val Val                       395                 400                 405               ACA ACC ACC GTG ATA TCG ATG CTG ATG CCG TTT TTT AAC GAC GTG   1339               Thr Thr Thr Val Ile Ser Met Leu Met Pro Phe Phe Asn Asp Val                       410                 415                 420               GTC GGG ATC TTA GGG GCG TTA GGG TTT TGG CCC TTG ACG GTT TAT   1384               Val Gly Ile Leu Gly Ala Leu Gly Phe Trp Pro Leu Thr Val Tyr                       425                 430                 435               TTT CCG GTG GAG ATG TAT ATT AAG CAG AGG AAG GTT GAG AAA TGG   1429               Phe Pro Val Glu Met Tyr Ile Lys Gln Arg Lys Val Glu Lys Trp                       440                 445                 450               AGC ACG AGA TGG GTG TGT TTA CAG ATG CTT AGT GTT GCT TGT CTT   1474               Ser Thr Arg Trp Val Cys Leu Gln Met Leu Ser Val Ala Cys Leu                       455                 460                 465               GTG ATC TCG GTG GTC GCC GGG GTT GGA TCA ATC GCC GGA GTG ATG   1519               Val Ile Ser Val Val Ala Gly Val Gly Ser Ile Ala Gly Val Met                       470                 475                 480               CTT GAT CTT AAG GTC TAT AAG CCA TTC AAG TCT ACA TAT   1558               Leu Asp Leu Lys Val Tyr Lys Pro Phe Lys Ser Thr Tyr                       485                 490               TGATGATTAT GGACCATGAA CAACAGAGAG AGTTGGTGTG TAAAGTTTAC   1608               CATTTCAAAG AAAACTCCAA AAATGTGTAT ATTGTATGTT GTTCTCATTT   1658               CGTATGGTCT CATCTTTGTA ATAAAATTTA AAACTTATGT TATAAATTAT   1708               AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AA   1740            
           [0016]    The DNA sequences of the invention identified with the help of the transformed yeast strains, e.g., sequences Seq. No. 1 and 2, can be introduced into plasmids and thereby be combined with steering elements for expression in eukaryotic cells (see Example 4). These steering elements are, on the one hand, transcription promoters, and, on the other hand, transcription terminators. Plasmids can be used to transform eukaryotic cells with the aim of expression of a translatable mRNA which makes possible the synthesis of an amino acid transporter in the cells or with the aim of expression of a non-translatable RNA, which prevents synthesis of an endogenous amino acid transporter in the cells. The expression of an RNA corresponding to the inventive sequences of plant amino acid transporters modifies the plant acid metabolism, as well as total nitrogen metabolism. The economic significance of this modification is obvious. Nitrogen is the nutrient mainly responsible for limiting growth. The viability of germ lines as well as germination capacity of seeds is directly dependent on the nitrogen content of storage tissue. The formation of high value food materials with a high protein content is dependent on a sufficient nitrogen supply. Nitrogen is transported essentially in the form of amino acids. An improvement in the delivery of amino acids to their harvested parts can therefore lead to an increase in yield of agricultural plants. The possibility of forcing the uptake of amino acid in individual organs allows the qualitative improvement of such organs, which, because of the demands of the utilization process, contain little nitrogen. An example is potatoes which are grown for the production of starch. Besides this, it is possible to modify the whole plant, by which the growth of individual tissues, for example, leaves, is slowed down, while the growth of the harvested parts is increased. For this, one can imagine a lengthening of the vegetative phase of crops, which leads to an increased formation of storage substances.  
           [0017]    Processes for the genetic modification of dicotyledonous and monocotyledonous plants are already known (see for example Gasser, C. S., Fraley, R. T., 1989, Science 244: 1293-1299; Potrykus, 191, Ann Rev Plant Mol Biol Plant Physiol 42: 205-225). For expression in plants the coding sequences must be coupled with the transcriptional regulatory elements. Such elements, called promoters, are known (EP 375091).  
           [0018]    Further, the coding regions must be provided with transcription termination signals with which they can be correctly transcribed. Such elements are also described (see Gielen et al., 1989, EMBO J 8: 23-29). The transcriptional start region can be either native and/or homologous or foreign and/or heterologous to the host plant. If desired, termination regions are interchangeable with one another. The DNA sequence of the transcription starting and termination regions can be prepared synthetically, obtained naturally, or can be a mixture of synthetic and natural DNA constituents. For introduction of foreign genes in higher plants, a large number of cloning vectors are available that include a replication signal for  E. coli  and a marker which allows for the selection of the transformed cells. Examples of such vectors are pBR 322, pUC-Series, M13 mp-Series, pACYC 184, etc. Depending on the method of introduction of the desired gene in the plants, other DNA sequences may be suitable. Should the Ti- or Ri-plasmid be used, e.g., for the transformation of the plant cell, then at least the right boundary, often, however, both the right and left boundary of the Ti- and Ri-Plasmid T-DNA, is attached, as a flanking region, to the gene being introduced. The use of T-DNA for the transformation of plant cells has been intensively researched and is well described in EP 120 516; Hoekama, In: The Binary Plant Vector System, Offset-drukkerij Kanters B. V. Alblasserdam (1985), Chapter V; Fraley, et al., Crit. Rev. Plant Sci., 4:1-46 and An et al. (1985) EMBO J. 4: 277-287. Once the introduced DNA is integrate in the genome, it is generally stable there and remains in the offspring of the original transformed cells. It normally contains a selection marker which induces resistance in the transformed plant cells against a biocide or antibiotic such as kanamycin, G 418, bleomycin, hygromycin or phosphinotricin, etc. The individual marker employed should therefore allow the selection of transformed cells from cells which lack the introduced DNA.  
           [0019]    For the introduction of DNA into a plant host cell, besides transformation using Agrobacteria, there are many other techniques available. These techniques include the fusion of protoplasts, microinjection of DNA and electroporation, as well as ballistic methods and virus infection. From the transformed plant material, whole plants can be regenerated in a suitable medium which contains antibiotics or biocides for selection. The resulting plants can then be tested for the presence of introduced DNA. No special demands are placed on the plasmids in injection and electroporation. Simple plasmids, such as, e.g., pUC-derivatives, can be used. Should whole plants be regenerated from such transformed cells, the presence of a selectable marker gene is necessary. The transformed cells grow within the plants in the usual manner (see also McCormick et al. (1986) Plant Cell Reports 5: 81-84). These plants can be grown normally and crossed with plants that possess the same transformed genes or different genes. The resulting hybrid individuals have the corresponding phenotypical properties.  
           [0020]    The DNA sequences of the invention can also be introduced in plasmids and thereby combined with steering elements for an expression in prokaryotic cells. The formation of a translatable RNA sequence of a eukaryotic amino acid transporter from bacteria, in spite of the considerable differences in the membrane structures of prokaryotes and eukaryotes, means that prokaryotes can now use a eukaryotic amino acid transporter with specificity for certain substrates. This makes possible the production of bacterial strains which could be used for studies of the properties of the transporter as well as its substrate.  
           [0021]    The invention also relates to bacteria that contain the plasmids of the invention.  
           [0022]    The DNA sequences of the invention can also be introduced in plasmids which allow mutagenesis or a sequence modification through recombination of DNA sequences in prokaryotic or eukaryotic systems. In this way, the specificity of the amino acid transporter can be modified. Thus, the specificity of the transporter can be changed.  
           [0023]    The invention also relates to derivatives or parts of plasmids that contain the DNA sequences of the invention and which can be used for the transformation of prokaryotic and eukaryotic cells.  
           [0024]    By using standard processes (see Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, NY, USA), base exchanges can be carried out or natural or synthetic sequences can be added. For binding DNA fragments with one another, adaptors or linkers can be introduced on the fragments. Further, manipulations can be carried which prepare suitable restriction cleavage sites or remove the excess DNA or restriction cleavage sites. Where insertions, deletions or substitutions such as, for example, transitions and transversions are desired, in vitro mutagenesis, primer repair, restrictions or ligations can be used. For methods of analysis, in general, a sequence analysis, restriction analysis and other biochemical molecular biological methods can be used. After each manipulation, the DNA sequence used can be cleaved and bound with another DNA sequence. Each plasmid sequence can be cloned in the same or different plasmids.  
           [0025]    Derivatives or parts of the DNA sequences and plasmids of the invention can also be used for the transformation of prokaryotic and eukaryotic cells. Further, the DNA sequences of the invention can be used according to standard processes for the isolation of similar sequences on the genome of plants of various species, which also code for amino acid or other oligosaccharide transporter molecules. With these sequence constructs, for the transformation of plant cells, can be prepared which modify the transport process in transgenic plants.  
           [0026]    In order to specify related DNA sequences, gene libraries must first be prepared which are representative of the content of genes of a plant type or for the expression of genes in a plant type. The former are genomic libraries, while the latter are cDNA libraries. From these, related sequences can be isolated using the DNA sequences of the invention as probes. Once the related gene has been identified and isolated, a determination of the sequence and an analysis of the properties of the proteins coded from this sequence is possible.  
           [0027]    In order to understand the examples forming the basis of this invention all the processes necessary for these tests and which are known per se will first of all be listed:  
           [0028]    1. Cloning process  
           [0029]    For cloning in  E. coli,  the vector pBluescriptSK (Short et al., 1988, Nucl Acids Res 16: 7583-7600) was used.  
           [0030]    For the transformation of yeasts, the vector pFL61 (Minet &amp; Lacroute, 1990, Curr Genet 18: 287-291) was used.  
           [0031]    For the plant transformation the gene constructs in the binary vector pBIN-Hyg were cloned.  
           [0032]    2. Bacterial and yeast strains  
           [0033]    For the pBluescriptSK vector as well as for PBinAR constructs, the  E. coli  strain XL1blue (Bullock et al., 1987, Biotechniques, 5, 376-378) was used.  
           [0034]    As a starting strain for the expression of the cDNA library in yeast, the yeast strain 22574d (Jauniaux et al., 1987 Eur J Biochem 164: 601-606) was used.  
           [0035]    The transformation of the plasmids in potato plants was carried out using  Agrobacterium tumefaciens  strain LBA4404 (Bevan (1984) Nucl. Acids Res 12: 8711-8720).  
           [0036]    3. Transformation of  Agrobacterium tumefaciens    
           [0037]    The transfer of the DNA in Agrobacteria was carried out by direct transformation by the method of Höfgen &amp; Willmitzer (1988, Nucleic Acids Res 16: 9877). The plasmid DNA of the transformed Agrobacterium was isolated in accordance with the method of Birnboim and Doly (1979) (Nucl Acids Res 7: 1513-1523) and was analyzed by gel electrophoresis after suitable restriction cleavage.  
           [0038]    4. Plant transformation  
           [0039]    Ten small leaves, wounded with a scalpel, of a sterile potato culture were placed in 10 ml of MS medium with 2% amino acid containing 30-50 μl of an  Agrobacterium tumefaciens  overnight culture grown under selection. After 3-5 minutes of gentle shaking, the leaves were laid out on MS medium of 1.6% glucose, 2 mg/l of zeatin ribose, 0.02 mg/l of naphthylacetic acid, 0.02 mg/l of gibberellic acid, 500 mg/l of claforan, 50 mg/l of kanamycin and 0.8% bacto agar. After incubation for one week at 25° C. and 3000 lux, the claforan concentration in the medium was reduced by half.  
           [0040]    Deposits  
           [0041]    The following plasmids and yeast strains were deposited at the Deutschen Sammlung von Mikroorganismen (DSM) in Braunschweig, Germany on 12.06.1992 (deposit number):  
                                                           Plasmid   pPPP1-20   (DSM 7129)           Plasmid   pBinPPP1-20   (DSM 7130)                      
 
           [0042]    Other features and advantages of the present invention will become apparent from the following description of the invention which refers to the accompanying drawings. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0043]    [0043]FIG. 1 shows the plasmid pPPP1-20 which contains the sequence Seq-ID No. 1. The finely drawn line corresponds to the sequence from pBluescriptSK. The thicker line represents the cDNA insert. The cleavage positions of the inserts are shown.  
         [0044]    [0044]FIG. 2 shows the uptake of  14 C-proline from the medium.  
         [0045]    no=time period of the uptake without competitor;  
         [0046]    proline=time period with fourfold excess of unlabeled proline;  
         [0047]    citrulline=time period with fourfold excess of unlabeled citrulline;  
         [0048]    GABA=time period with fourfold excess of gamma-aminobutyric acid;  
         [0049]    time=time in seconds;  
         [0050]    cpm=decays counted per minute.  
         [0051]    [0051]FIG. 3 shows the plasmid pAAP2 which contains the sequence Seq-ID No. 2. The finely drawn line corresponds to the sequence from pBluescriptSK. The thicker line represents the cDNA insert. The cleavage positions of the inserts are shown.  
         [0052]    [0052]FIG. 4 shows a competition experiment with the yeast line 22574d::AAP2. In this experiment, the uptake of  14 C-labeled L-proline from the medium in the presence of a fourfold excess of other amino acids or their analogues is measured. Besides the standard abbreviations for amino acids in the three letter code, the following are also used:  
         [0053]    Cit=citrulline;  
         [0054]    D-Pro=D-proline;  
         [0055]    OH-Pro=hydroxyproline; and  
         [0056]    AC2=azetidine-2-carboxylic acid.  
         [0057]    [0057]FIG. 5 shows a competition experiment with the yeast line JT16::AAP2. In this experiment, the uptake of  14 C labeled L-histidine from the medium in the presence of a tenfold excess of other amino acids or their analogues is measured.  
         [0058]    Besides the standard abbreviations for amino acids in the three letter code, the following are also used:  
         [0059]    Cit=citrulline;  
         [0060]    Orn=ornithine;  
         [0061]    Can=canavanine; and  
         [0062]    NH4=ammonium. 
     
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS  
       [0063]    The following examples describe the cloning and identification, as well as the function and use of a plant amino acid transporter.  
       EXAMPLE 1  
     Cloning of the cDNA of a Plant Amino Acid Transporter  
       [0064]    For complementation of the proline transport mutation of the yeast strain 22574d (Jauniaux et al., 1987, Eur J Biochem 164: 601-606) and/or the histidine synthesis and transport mutation of the strain JT16 (Tanaka &amp; Fink 1985, Gene 38: 205-214), a cDNA of young germ lines from  Arabidopsis thaliana  (two leaf stage) in the yeast expression vector pFL61 (Minet &amp; Lacroute), 1990 Curr Genet 18: 287-291) which had been made available by Minet (Minet et al., 1992, Plant J 2: 417-422) was used. Around 1 μg of the vector with the cDNA-insert was transformed in the yeast strain 22574d and/or JT16 by the method of Dohmen et al. (1991, Yeast 7: 691-692). Yeast transformands, which could grow in media with 4 mM proline as the sole nitrogen source or in media with 6 mM histidine, were propagated. From the lines plasmid-DNA was prepared by standard methods. Clones that could complement the particular mutation contained plasmids with similar restriction type of the cDNA insert. These varied in size between 1.6 and 1.7 kb.  
       EXAMPLE 2  
     Sequence Analysis of the cDNA Insert of the Plasmid pFL61-ppp1-20  
       [0065]    From a yeast line PPP1-20, obtained in a similar manner to example 1, which, in spite of the 22574d mutation, could grow with proline as the only nitrogen source, the plasmid pFL61-ppp1-20 was isolated. Its cDNA insert was prepared as a NotI fragment and cloned in the vector pBluescriptSK. In this way, the plasmid pPPP1-20 was obtained (see FIG. 1). Using synthetic oligonucleotides, the insert was sequenced by the method of Sanger et al. (1977, Proc Natl Acad Sci USA 74:5463-5467). The sequence is given above (SEQ ID No. 1).  
         [0066]    In a similar way, from a yeast line that, in spite of the his4/hip1 double mutation, could be grown in a medium with histidine addition, the plasmid pFL61-aap2 was isolated whose insert was also cloned as a NotI fragment in pBluescriptSK. The resulting plasmid pAAP2 was sequenced and the sequence (SEQ ID No. 2) is given above. The plasmid pAAP2 has a similar structure to pPPP1-20 (see FIG. 1), but instead of the insert SEQ ID No. 1, carries the insert SEQ ID No. 2 (see FIG. 3).  
       EXAMPLE 3  
     Uptake Studies with  14 C-labeled Protein into the Yeast Line PPP1-20 and AAP2  
       [0067]    The yeast lines 22574d::PPP1-20 and 22574d::AAP2 that were obtained in a similar manner to Example 1 were grown in liquid medium until the culture reached the logarithmic phase. After centrifuging the culture, the cells are washed and taken up in 100 mm tris/HCl pH 4.5, 2 mM MgCl 2  and 0.6M sorbitol. Around 100 μL of the suspension was added to a solution of 0.5 mM L-proline plus 1 μCi  14 C labeled L-proline in 100 μL of the same buffer. The uptake of the labeled amino acid was measured by the process described by Cirillo (1989, Meth Enzymol 174: 617-622). The uptake of the labeled amino acid was compared, on the one hand, in co-incubation with protein modifying substance diethyl pyrocarbonate which is an inhibitor of the amino acid transport in membrane vesicles from Beta vulgaris, and, on the other hand, in co-incubation with other protein modifying substances. The calculated reduction is shown in Tables I and/or III. A competition experiment in which the specificity of the transporter could be read off with various amino acids and analogues is shown in Table II for PPP1-20 and in FIG. 4 for AAP2. An analogous experiment in which a competition for histidine uptake in the line JT16::AAP2 was tested is described in Example 5. The time period for PPP1-20 is shown in FIG. 2.  
       EXAMPLE 4  
     Transformation of Plants with a Construct for Overexpression of the Coding Region of Amino Acid Transporters  
       [0068]    From the plasmid pPPP1-20 that contains the cDNA for the amino acid transporter from Arabidopsis, an internal fragment of the insert was isolated after BamHI cleavage and cloned in the BamHI cleavage position from pAJ that was first linearized with the enzyme BamHI. Then the cDNA was prepared as the EcoRI/HindIII fragment from pA7 and cloned in the vector PBIN-HYG. After transformation by Agrobacteria, this was inserted for infection of leaf segments of tobacco and potato.  
         [0069]    Ten independently obtained transformands in which the presence of the intact non-rearranged chimeric gene was demonstrated using Southern blot analysis were tested for modifications of amino acid and nitrogen content. Besides this, amino acid synthesis, photosynthesis rate and transportation were tested.  
       EXAMPLE 5  
     Studies in the Uptake of  14 C-labeled Histidine in the Yeast Line AAP2  
       [0070]    The yeast line JT16::AAP2, obtained in a similar manner to Example 1, was grown in liquid medium until the culture reached the logarithmic phase. After centrifuging the culture, the cells were washed and taken up in 10 mm tris/HCl pH 4.5, 2 mm MgCl 2  and 0.6M sorbitol. Around 100 ml of the suspension was added to a solution of 0.5 mm L-histidine plus 1 μCi  14 C-labeled L-histidine in 100 μL of the same buffer. The uptake of the labeled amino acid was measured according to the method described by von Cirillo (1989, Meth Enzymol 174: 617-622). The uptake of the labeled amino acid was compared in a competition experiment with that from different amino acids and analogues in tenfold excess. The relationships are shown in FIG. 5.  
                                   TABLE I                           Inhibition of the amino acid transport in       22574d::PPP1-20 - yeast strains by protein modifying substances                % of transport           without inhibitor                    0.1 mM DEPC   65       (diethyl pyrocarbonate)        10 μM CCCP   &lt;3       (Carbonyl cyanide m-chlorophenylhydrazone)        10 μM 2, 4 DNP   &lt;3       (Dinitrophenol)         1 mM sodium arsenate   35        10 μM antimycin A   29       500 μM PCMBS   78       (p-chloromercuribenzenesulfonic acid)                  
 
         [0071]    [0071]                                     TABLE II                           Competition by one, fourfold and tenfold excess of amino       acids and analogues in 22574d::PPP1-20 - yeast strain                Excess % remaining                       transport activity:   1 x   4 x   10 x                       glutamic acid   64   27   30           aspartic acid   78       27           lysine   86       83           histidine   81   79   58           arginine   85   88   74           threonine   —   50   —           L-proline   49   21   14           D-proline   98       95           3,4-di-OH proline   86       49           azetidine-           2-carboxylic acid   91       48           OH-proline   81       45           valine   —   77   47           isoleucine   —   67   —           asparagine   64       57           glutamine   —   27   —           serine   53       18           cysteine   —   21   —           methionine   28        8           glycine   69       16           alanine   55   29   23           leucine   —       —           tyrosine   —       —           tryptophan   82   71   48           phenylalanine   45       16           citrulline       44           gamma-aminobutyric acid       90                        
         [0072]    [0072]                                       TABLE III                           Inhibition of the amino acid transports in       JT16::AAP2 - yeast strain by protein modifying substances                % of transport without inhibitor                            1 mM DEPC   3.1 ± 1.6           (Diethyl pyrocarbonate)           10 μM CCCP   15.6 ± 2.1            (Carbonyl cyanide           m-chlorophenylhydrazone)           10 μM 2,4 DNP           (Dinitrophenol)   7.6 ± 1.6                        
         [0073]    Although the present invention has been described in relation to particular embodiments thereof, many other variations and modifications and other uses will become apparent to those skilled in the art. Therefore, the present invention is to be limited not by the specific disclosure herein, but only by the appended claims.   
     
       
       
         1 
         
           
             
4 
 
           
           
             
               1685 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
             
               Arabidopsis thaliano  
             
             
               CDS  
                57..1511  
                /note= “amino acid transporter”
 
             
              1 

CTTAAAACAT TTATTTTATC TTCTTCTTGT TCTCTCTTTC TCTTTCTCTC ATCACT         56 

ATG AAG AGT TTC AAC ACA GAA GGA CAC AAC CAC TCC ACG GCG GAA TCC      104 
Met Lys Ser Phe Asn Thr Glu Gly His Asn His Ser Thr Ala Glu Ser 
  1               5                  10                  15 

GGC GAT GCC TAC ACC GTG TCG GAC CCG ACA AAG AAC GTC GAT GAA GAT      152 
Gly Asp Ala Tyr Thr Val Ser Asp Pro Thr Lys Asn Val Asp Glu Asp 
             20                  25                  30 

GGT CGA GAG AAG CGT ACC GGG ACG TGG CTT ACG GCG AGT GCG CAT ATT      200 
Gly Arg Glu Lys Arg Thr Gly Thr Trp Leu Thr Ala Ser Ala His Ile 
         35                  40                  45 

ATC ACG GCG GTG ATA GGC TCC GGA GTG TTG TCT TTA GCA TGG GCT ATA      248 
Ile Thr Ala Val Ile Gly Ser Gly Val Leu Ser Leu Ala Trp Ala Ile 
     50                  55                  60 

GCT CAG CTT GGT TGG ATC GCA GGG ACA TCG ATC TTA CTC ATT TTC TCG      296 
Ala Gln Leu Gly Trp Ile Ala Gly Thr Ser Ile Leu Leu Ile Phe Ser 
 65                  70                  75                  80 

TTC ATT ACT TAC TTC ACC TCC ACC ATG CTT GCC GAT TGC TAC CGT GCG      344 
Phe Ile Thr Tyr Phe Thr Ser Thr Met Leu Ala Asp Cys Tyr Arg Ala 
                 85                  90                  95 

CCG GAT CCC GTC ACC GGA AAA CGG AAT TAC ACT TAC ATG GAC GTT GTT      392 
Pro Asp Pro Val Thr Gly Lys Arg Asn Tyr Thr Tyr Met Asp Val Val 
            100                 105                 110 

CGA TCT TAC CTC GGT GGT AGG AAA GTG CAG CTC TGT GGA GTG GCA CAA      440 
Arg Ser Tyr Leu Gly Gly Arg Lys Val Gln Leu Cys Gly Val Ala Gln 
        115                 120                 125 

TAT GGG AAT CTG ATT GGG GTC ACT GTT GGT TAC ACC ATC ACT GCT TCT      488 
Tyr Gly Asn Leu Ile Gly Val Thr Val Gly Tyr Thr Ile Thr Ala Ser 
    130                 135                 140 

ATT AGT TTG GTA GCG GTA GGG AAA TCG AAC TGC TTC CAC GAT AAA GGG      536 
Ile Ser Leu Val Ala Val Gly Lys Ser Asn Cys Phe His Asp Lys Gly 
145                 150                 155                 160 

CAC ACT GCG GAT TGT ACT ATA TCG AAT TAT CCG TAT ATG GCG GTT TTT      584 
His Thr Ala Asp Cys Thr Ile Ser Asn Tyr Pro Tyr Met Ala Val Phe 
                165                 170                 175 

GGT ATC ATT CAA GTT ATT CTT AGC CAG ATC CCA AAT TTC CAC AAG CTC      632 
Gly Ile Ile Gln Val Ile Leu Ser Gln Ile Pro Asn Phe His Lys Leu 
            180                 185                 190 

TCT TTT CTT TCC ATT ATG GCC GCA GTC ATG TCC TTT ACT TAT GCA ACT      680 
Ser Phe Leu Ser Ile Met Ala Ala Val Met Ser Phe Thr Tyr Ala Thr 
        195                 200                 205 

ATT GGA ATC GGT CTA GCC ATC GCA ACC GTC GCA GGT GGG AAA GTG GGT      728 
Ile Gly Ile Gly Leu Ala Ile Ala Thr Val Ala Gly Gly Lys Val Gly 
    210                 215                 220 

AAG ACG AGT ATG ACG GGC ACA GCG GTT GGA GTA GAT GTA ACC GCA GCT      776 
Lys Thr Ser Met Thr Gly Thr Ala Val Gly Val Asp Val Thr Ala Ala 
225                 230                 235                 240 

CAA AAG ATA TGG AGA TCG TTT CAA GCG GTT GGG GAC ATA GCG TTC GCC      824 
Gln Lys Ile Trp Arg Ser Phe Gln Ala Val Gly Asp Ile Ala Phe Ala 
                245                 250                 255 

TAT GCT TAT GCC ACG GTT CTC ATC GAG ATT CAG GAT ACA CTA AGA TCT      872 
Tyr Ala Tyr Ala Thr Val Leu Ile Glu Ile Gln Asp Thr Leu Arg Ser 
            260                 265                 270 

AGC CCA GCT GAG AAC AAA GCC ATG AAA AGA GCA AGT CTT GTG GGA GTA      920 
Ser Pro Ala Glu Asn Lys Ala Met Lys Arg Ala Ser Leu Val Gly Val 
        275                 280                 285 

TCA ACC ACC ACT TTT TTC TAC ATC TTA TGT GGA TGC ATC GGC TAT GCT      968 
Ser Thr Thr Thr Phe Phe Tyr Ile Leu Cys Gly Cys Ile Gly Tyr Ala 
    290                 295                 300 

GCA TTT GGA AAC AAT GCC CCT GGA GAT TTC CTC ACA GAT TTC GGG TTT     1016 
Ala Phe Gly Asn Asn Ala Pro Gly Asp Phe Leu Thr Asp Phe Gly Phe 
305                 310                 315                 320 

TTC GAG CCC TTT TGG CTC ATT GAC TTT GCA AAC GCT TGC ATC GCT GTC     1064 
Phe Glu Pro Phe Trp Leu Ile Asp Phe Ala Asn Ala Cys Ile Ala Val 
                325                 330                 335 

CAC CTT ATT GGT GCC TAT CAG GTG TTC GCG CAG CCG ATA TTC CAG TTT     1112 
His Leu Ile Gly Ala Tyr Gln Val Phe Ala Gln Pro Ile Phe Gln Phe 
            340                 345                 350 

GTT GAG AAA AAA TGC AAC AGA AAC TAT CCA GAC AAC AAG TTC ATC ACT     1160 
Val Glu Lys Lys Cys Asn Arg Asn Tyr Pro Asp Asn Lys Phe Ile Thr 
        355                 360                 365 

TCT GAA TAT TCA GTA AAC GTA CCT TTC CTT GGA AAA TTC AAC ATT AGC     1208 
Ser Glu Tyr Ser Val Asn Val Pro Phe Leu Gly Lys Phe Asn Ile Ser 
    370                 375                 380 

CTC TTC AGA TTG GTG TGG AGG ACA GCT TAT GTG GTT ATA ACC ACT GTT     1256 
Leu Phe Arg Leu Val Trp Arg Thr Ala Tyr Val Val Ile Thr Thr Val 
385                 390                 395                 400 

GTA GCT ATG ATA TTC CCT TTC TTC AAC GCG ATC TTA GGT CTT ATC GGA     1304 
Val Ala Met Ile Phe Pro Phe Phe Asn Ala Ile Leu Gly Leu Ile Gly 
                405                 410                 415 

GCA GCT TCC TTC TGG CCT TTA ACG GTT TAT TTC CCT GTG GAG ATG CAC     1352 
Ala Ala Ser Phe Trp Pro Leu Thr Val Tyr Phe Pro Val Glu Met His 
            420                 425                 430 

ATT GCA CAA ACC AAG ATT AAG AAG TAC TCT GCT AGA TGG ATT GCG CTG     1400 
Ile Ala Gln Thr Lys Ile Lys Lys Tyr Ser Ala Arg Trp Ile Ala Leu 
        435                 440                 445 

AAA ACG ATG TGC TAT GTT TGC TTG ATC GTC TCG CTC TTA GCT GCA GCC     1448 
Lys Thr Met Cys Tyr Val Cys Leu Ile Val Ser Leu Leu Ala Ala Ala 
    450                 455                 460 

GGA TCC ATC GCA GGA CTT ATA AGT AGT GTC AAA ACC TAC AAG CCC TTC     1496 
Gly Ser Ile Ala Gly Leu Ile Ser Ser Val Lys Thr Tyr Lys Pro Phe 
465                 470                 475                 480 

CGG ACT ATG CAT GAG TGAGTTTGAG ATCCTCAAGA GAGTCAAAAA TATATGTAGT     1551 
Arg Thr Met His Glu 
                485 

AGTTTGGTCT TTCTGTTAAA CTATCTGGTG TCTAAATCCA ATGAGAATGC TTTATTGC     1611 

AAACTTCATG AATCTCTCTG TATCTACATC TTTCAATCTA ATACATATGA GCTCTTCC     1671 

AAAAAAAAAA AAAA                                                     1685 

 
           
           
             
               485 amino acids  
               amino acid  
               linear  
             
             
               protein  
             
              2 

Met Lys Ser Phe Asn Thr Glu Gly His Asn His Ser Thr Ala Glu Ser 
  1               5                  10                  15 

Gly Asp Ala Tyr Thr Val Ser Asp Pro Thr Lys Asn Val Asp Glu Asp 
             20                  25                  30 

Gly Arg Glu Lys Arg Thr Gly Thr Trp Leu Thr Ala Ser Ala His Ile 
         35                  40                  45 

Ile Thr Ala Val Ile Gly Ser Gly Val Leu Ser Leu Ala Trp Ala Ile 
     50                  55                  60 

Ala Gln Leu Gly Trp Ile Ala Gly Thr Ser Ile Leu Leu Ile Phe Ser 
 65                  70                  75                  80 

Phe Ile Thr Tyr Phe Thr Ser Thr Met Leu Ala Asp Cys Tyr Arg Ala 
                 85                  90                  95 

Pro Asp Pro Val Thr Gly Lys Arg Asn Tyr Thr Tyr Met Asp Val Val 
            100                 105                 110 

Arg Ser Tyr Leu Gly Gly Arg Lys Val Gln Leu Cys Gly Val Ala Gln 
        115                 120                 125 

Tyr Gly Asn Leu Ile Gly Val Thr Val Gly Tyr Thr Ile Thr Ala Ser 
    130                 135                 140 

Ile Ser Leu Val Ala Val Gly Lys Ser Asn Cys Phe His Asp Lys Gly 
145                 150                 155                 160 

His Thr Ala Asp Cys Thr Ile Ser Asn Tyr Pro Tyr Met Ala Val Phe 
                165                 170                 175 

Gly Ile Ile Gln Val Ile Leu Ser Gln Ile Pro Asn Phe His Lys Leu 
            180                 185                 190 

Ser Phe Leu Ser Ile Met Ala Ala Val Met Ser Phe Thr Tyr Ala Thr 
        195                 200                 205 

Ile Gly Ile Gly Leu Ala Ile Ala Thr Val Ala Gly Gly Lys Val Gly 
    210                 215                 220 

Lys Thr Ser Met Thr Gly Thr Ala Val Gly Val Asp Val Thr Ala Ala 
225                 230                 235                 240 

Gln Lys Ile Trp Arg Ser Phe Gln Ala Val Gly Asp Ile Ala Phe Ala 
                245                 250                 255 

Tyr Ala Tyr Ala Thr Val Leu Ile Glu Ile Gln Asp Thr Leu Arg Ser 
            260                 265                 270 

Ser Pro Ala Glu Asn Lys Ala Met Lys Arg Ala Ser Leu Val Gly Val 
        275                 280                 285 

Ser Thr Thr Thr Phe Phe Tyr Ile Leu Cys Gly Cys Ile Gly Tyr Ala 
    290                 295                 300 

Ala Phe Gly Asn Asn Ala Pro Gly Asp Phe Leu Thr Asp Phe Gly Phe 
305                 310                 315                 320 

Phe Glu Pro Phe Trp Leu Ile Asp Phe Ala Asn Ala Cys Ile Ala Val 
                325                 330                 335 

His Leu Ile Gly Ala Tyr Gln Val Phe Ala Gln Pro Ile Phe Gln Phe 
            340                 345                 350 

Val Glu Lys Lys Cys Asn Arg Asn Tyr Pro Asp Asn Lys Phe Ile Thr 
        355                 360                 365 

Ser Glu Tyr Ser Val Asn Val Pro Phe Leu Gly Lys Phe Asn Ile Ser 
    370                 375                 380 

Leu Phe Arg Leu Val Trp Arg Thr Ala Tyr Val Val Ile Thr Thr Val 
385                 390                 395                 400 

Val Ala Met Ile Phe Pro Phe Phe Asn Ala Ile Leu Gly Leu Ile Gly 
                405                 410                 415 

Ala Ala Ser Phe Trp Pro Leu Thr Val Tyr Phe Pro Val Glu Met His 
            420                 425                 430 

Ile Ala Gln Thr Lys Ile Lys Lys Tyr Ser Ala Arg Trp Ile Ala Leu 
        435                 440                 445 

Lys Thr Met Cys Tyr Val Cys Leu Ile Val Ser Leu Leu Ala Ala Ala 
    450                 455                 460 

Gly Ser Ile Ala Gly Leu Ile Ser Ser Val Lys Thr Tyr Lys Pro Phe 
465                 470                 475                 480 

Arg Thr Met His Glu 
                485 

 
           
           
             
               1740 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
             
               Arabidopsis thaliana  
             
             
               CDS  
                80..1558  
                /product= “amino acid transporter”
 
             
              3 

CTATTTTATA ATTCCTCTTC TTTTTGTTCA TAGCTTTGTA ATTATAGTCT TATTTCTCTT     60 

TAAGGCTCAA TAAGAGGAG ATG GGT GAA ACC GCT GCC GCC AAT AAC CAC CGT     112 

                     Met Gly Glu Thr Ala Ala Ala Asn Asn His Arg 
                       1               5                  10 

CAC CAC CAC CAT CAC GGC CAC CAG GTC TTT GAC GTG GCC AGC CAC GAT      160 
His His His His His Gly His Gln Val Phe Asp Val Ala Ser His Asp 
             15                  20                  25 

TTC GTC CCT CCA CAA CCG GCT TTT AAA TGC TTC GAT GAT GAT GGC CGC      208 
Phe Val Pro Pro Gln Pro Ala Phe Lys Cys Phe Asp Asp Asp Gly Arg 
         30                  35                  40 

CTC AAA AGA ACT GGG ACT GTT TGG ACC GCG AGC GCT CAT ATA ATA ACT      256 
Leu Lys Arg Thr Gly Thr Val Trp Thr Ala Ser Ala His Ile Ile Thr 
     45                  50                  55 

GCG GTT ATC GGA TCC GGC GTT TTG TCA TTG GCG TGG GCG ATT GCA CAG      304 
Ala Val Ile Gly Ser Gly Val Leu Ser Leu Ala Trp Ala Ile Ala Gln 
 60                  65                  70                  75 

CTC GGA TGG ATC GCT GGC CCT GCT GTG ATG CTA TTG TTC TCT CTT GTT      352 
Leu Gly Trp Ile Ala Gly Pro Ala Val Met Leu Leu Phe Ser Leu Val 
                 80                  85                  90 

ACT CTT TAC TCC TCC ACA CTT CTT AGC GAC TGC TAC AGA ACC GGC GAT      400 
Thr Leu Tyr Ser Ser Thr Leu Leu Ser Asp Cys Tyr Arg Thr Gly Asp 
             95                 100                 105 

GCA GTG TCT GGC AAG AGA AAC TAC ACT TAC ATG GAT GCC GTT CGA TCA      448 
Ala Val Ser Gly Lys Arg Asn Tyr Thr Tyr Met Asp Ala Val Arg Ser 
        110                 115                 120 

ATT CTC GGT GGG TTC AAG TTC AAG ATT TGT GGG TTG ATT CAA TAC TTG      496 
Ile Leu Gly Gly Phe Lys Phe Lys Ile Cys Gly Leu Ile Gln Tyr Leu 
    125                 130                 135 

AAT CTC TTT GGT ATC GCA ATT GGA TAC ACG ATA GCA GCT TCC ATA AGC      544 
Asn Leu Phe Gly Ile Ala Ile Gly Tyr Thr Ile Ala Ala Ser Ile Ser 
140                 145                 150                 155 

ATG ATG GCG ATC AAG AGA TCC AAC TGC TTC CAC AAG AGT GGA GGA AAA      592 
Met Met Ala Ile Lys Arg Ser Asn Cys Phe His Lys Ser Gly Gly Lys 
                160                 165                 170 

GAC CCA TGT CAC ATG TCC AGT AAT CCT TAC ATG ATC GTA TTT GGT GTG      640 
Asp Pro Cys His Met Ser Ser Asn Pro Tyr Met Ile Val Phe Gly Val 
            175                 180                 185 

GCA GAG ATC TTG CTC TCT CAG GTT CCT GAT TTC GAT CAG ATT TGG TGG      688 
Ala Glu Ile Leu Leu Ser Gln Val Pro Asp Phe Asp Gln Ile Trp Trp 
        190                 195                 200 

ATC TCC ATT GTT GCA GCT GTT ATG TCC TTC ACT TAC TCT GCC ATT GGT      736 
Ile Ser Ile Val Ala Ala Val Met Ser Phe Thr Tyr Ser Ala Ile Gly 
    205                 210                 215 

CTA GCT CTT GGA ATC GTT CAA GTT GCA GCG AAT GGA GTT TTC AAA GGA      784 
Leu Ala Leu Gly Ile Val Gln Val Ala Ala Asn Gly Val Phe Lys Gly 
220                 225                 230                 235 

AGT CTC ACT GGA ATA AGC ATC GGA ACA GTG ACT CAA ACA CAG AAG ATA      832 
Ser Leu Thr Gly Ile Ser Ile Gly Thr Val Thr Gln Thr Gln Lys Ile 
                240                 245                 250 

TGG AGA ACC TTC CAA GCA CTT GGA GAC ATT GCC TTT GCG TAC TCA TAC      880 
Trp Arg Thr Phe Gln Ala Leu Gly Asp Ile Ala Phe Ala Tyr Ser Tyr 
            255                 260                 265 

TCT GTT GTC CTA ATC GAG ATT CAG GAT ACT GTA AGA TCC CCA CCG GCG      928 
Ser Val Val Leu Ile Glu Ile Gln Asp Thr Val Arg Ser Pro Pro Ala 
        270                 275                 280 

GAA TCG AAA ACG ATG AAG AAA GCA ACA AAA ATC AGT ATT GCC GTC ACA      976 
Glu Ser Lys Thr Met Lys Lys Ala Thr Lys Ile Ser Ile Ala Val Thr 
    285                 290                 295 

ACT ATC TTC TAC ATG CTA TGT GGC TCA ATG GGT TAT GCC GCT TTT GGA     1024 
Thr Ile Phe Tyr Met Leu Cys Gly Ser Met Gly Tyr Ala Ala Phe Gly 
300                 305                 310                 315 

GAT GCA GCA CCG GGA AAC CTC CTC ACC GGT TTT GGA TTC TAC AAC CCG     1072 
Asp Ala Ala Pro Gly Asn Leu Leu Thr Gly Phe Gly Phe Tyr Asn Pro 
                320                 325                 330 

TTT TGG CTC CTT GAC ATA GCT AAC GCC GCC ATT GTT GTC CAC CTC GTT     1120 
Phe Trp Leu Leu Asp Ile Ala Asn Ala Ala Ile Val Val His Leu Val 
            335                 340                 345 

GGA GCT TAC CAA GTC TTT GCT CAG CCC ATC TTT GCC TTT ATT GAA AAA     1168 
Gly Ala Tyr Gln Val Phe Ala Gln Pro Ile Phe Ala Phe Ile Glu Lys 
        350                 355                 360 

TCA GTC GCA GAG AGA TAT CCA GAC AAT GAC TTC CTC AGC AAG GAA TTT     1216 
Ser Val Ala Glu Arg Tyr Pro Asp Asn Asp Phe Leu Ser Lys Glu Phe 
    365                 370                 375 

GAA ATC AGA ATC CCC GGA TTT AAG TCT CCT TAC AAA GTA AAC GTT TTC     1264 
Glu Ile Arg Ile Pro Gly Phe Lys Ser Pro Tyr Lys Val Asn Val Phe 
380                 385                 390                 395 

AGG ATG GTT TAC AGG AGT GGC TTT GTC GTT ACA ACC ACC GTG ATA TCG     1312 
Arg Met Val Tyr Arg Ser Gly Phe Val Val Thr Thr Thr Val Ile Ser 
                400                 405                 410 

ATG CTG ATG CCG TTT TTT AAC GAC GTG GTC GGG ATC TTA GGG GCG TTA     1360 
Met Leu Met Pro Phe Phe Asn Asp Val Val Gly Ile Leu Gly Ala Leu 
            415                 420                 425 

GGG TTT TGG CCC TTG ACG GTT TAT TTT CCG GTG GAG ATG TAT ATT AAG     1408 
Gly Phe Trp Pro Leu Thr Val Tyr Phe Pro Val Glu Met Tyr Ile Lys 
        430                 435                 440 

CAG AGG AAG GTT GAG AAA TGG AGC ACG AGA TGG GTG TGT TTA CAG ATG     1456 
Gln Arg Lys Val Glu Lys Trp Ser Thr Arg Trp Val Cys Leu Gln Met 
    445                 450                 455 

CTT AGT GTT GCT TGT CTT GTG ATC TCG GTG GTC GCC GGG GTT GGA TCA     1504 
Leu Ser Val Ala Cys Leu Val Ile Ser Val Val Ala Gly Val Gly Ser 
460                 465                 470                 475 

ATC GCC GGA GTG ATG CTT GAT CTT AAG GTC TAT AAG CCA TTC AAG TCT     1552 
Ile Ala Gly Val Met Leu Asp Leu Lys Val Tyr Lys Pro Phe Lys Ser 
                480                 485                 490 

ACA TAT TGATGATTAT GGACCATGAA CAACAGAGAG AGTTGGTGTG TAAAGTTTAC      1608 
Thr Tyr 
CATTTCAAAG AAAACTCCAA AAATGTGTAT ATTGTATGTT GTTCTCATTT CGTATGGT     1668 

CATCTTTGTA ATAAAATTTA AAACTTATGT TATAAATTAT AAAAAAAAAA AAAAAAAA     1728 

AAAAAAAAAA AA                                                       1740 

 
           
           
             
               493 amino acids  
               amino acid  
               linear  
             
             
               protein  
             
              4 

Met Gly Glu Thr Ala Ala Ala Asn Asn His Arg His His His His His 
  1               5                  10                  15 

Gly His Gln Val Phe Asp Val Ala Ser His Asp Phe Val Pro Pro Gln 
             20                  25                  30 

Pro Ala Phe Lys Cys Phe Asp Asp Asp Gly Arg Leu Lys Arg Thr Gly 
         35                  40                  45 

Thr Val Trp Thr Ala Ser Ala His Ile Ile Thr Ala Val Ile Gly Ser 
     50                  55                  60 

Gly Val Leu Ser Leu Ala Trp Ala Ile Ala Gln Leu Gly Trp Ile Ala 
 65                  70                  75                  80 

Gly Pro Ala Val Met Leu Leu Phe Ser Leu Val Thr Leu Tyr Ser Ser 
                 85                  90                  95 

Thr Leu Leu Ser Asp Cys Tyr Arg Thr Gly Asp Ala Val Ser Gly Lys 
            100                 105                 110 

Arg Asn Tyr Thr Tyr Met Asp Ala Val Arg Ser Ile Leu Gly Gly Phe 
        115                 120                 125 

Lys Phe Lys Ile Cys Gly Leu Ile Gln Tyr Leu Asn Leu Phe Gly Ile 
    130                 135                 140 

Ala Ile Gly Tyr Thr Ile Ala Ala Ser Ile Ser Met Met Ala Ile Lys 
145                 150                 155                 160 

Arg Ser Asn Cys Phe His Lys Ser Gly Gly Lys Asp Pro Cys His Met 
                165                 170                 175 

Ser Ser Asn Pro Tyr Met Ile Val Phe Gly Val Ala Glu Ile Leu Leu 
            180                 185                 190 

Ser Gln Val Pro Asp Phe Asp Gln Ile Trp Trp Ile Ser Ile Val Ala 
        195                 200                 205 

Ala Val Met Ser Phe Thr Tyr Ser Ala Ile Gly Leu Ala Leu Gly Ile 
    210                 215                 220 

Val Gln Val Ala Ala Asn Gly Val Phe Lys Gly Ser Leu Thr Gly Ile 
225                 230                 235                 240 

Ser Ile Gly Thr Val Thr Gln Thr Gln Lys Ile Trp Arg Thr Phe Gln 
                245                 250                 255 

Ala Leu Gly Asp Ile Ala Phe Ala Tyr Ser Tyr Ser Val Val Leu Ile 
            260                 265                 270 

Glu Ile Gln Asp Thr Val Arg Ser Pro Pro Ala Glu Ser Lys Thr Met 
        275                 280                 285 

Lys Lys Ala Thr Lys Ile Ser Ile Ala Val Thr Thr Ile Phe Tyr Met 
    290                 295                 300 

Leu Cys Gly Ser Met Gly Tyr Ala Ala Phe Gly Asp Ala Ala Pro Gly 
305                 310                 315                 320 

Asn Leu Leu Thr Gly Phe Gly Phe Tyr Asn Pro Phe Trp Leu Leu Asp 
                325                 330                 335 

Ile Ala Asn Ala Ala Ile Val Val His Leu Val Gly Ala Tyr Gln Val 
            340                 345                 350 

Phe Ala Gln Pro Ile Phe Ala Phe Ile Glu Lys Ser Val Ala Glu Arg 
        355                 360                 365 

Tyr Pro Asp Asn Asp Phe Leu Ser Lys Glu Phe Glu Ile Arg Ile Pro 
    370                 375                 380 

Gly Phe Lys Ser Pro Tyr Lys Val Asn Val Phe Arg Met Val Tyr Arg 
385                 390                 395                 400 

Ser Gly Phe Val Val Thr Thr Thr Val Ile Ser Met Leu Met Pro Phe 
                405                 410                 415 

Phe Asn Asp Val Val Gly Ile Leu Gly Ala Leu Gly Phe Trp Pro Leu 
            420                 425                 430 

Thr Val Tyr Phe Pro Val Glu Met Tyr Ile Lys Gln Arg Lys Val Glu 
        435                 440                 445 

Lys Trp Ser Thr Arg Trp Val Cys Leu Gln Met Leu Ser Val Ala Cys 
    450                 455                 460 

Leu Val Ile Ser Val Val Ala Gly Val Gly Ser Ile Ala Gly Val Met 
465                 470                 475                 480 

Leu Asp Leu Lys Val Tyr Lys Pro Phe Lys Ser Thr Tyr 
                485                 490