Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide encodes an inhibitory rna molecule
Reexamination Certificate
2001-05-14
2004-10-26
Fox, David T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide encodes an inhibitory rna molecule
C800S278000, C800S298000, C536S023100, C536S023600, C435S252300, C435S254200, C435S320100, C435S419000
Reexamination Certificate
active
06809233
ABSTRACT:
BACKGROUND OF THE INVENTION
Field of the Invention
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.
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.
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.
Corresponding complementation studies have been carried out with the eukaryotic yeast,
Saccharomyces cerevisiae
. Tanaka & 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.
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 & 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.
The transporter molecule or gene for plant transporter proteins is not known.
SUMMARY OF THE INVENTION
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.
Under the term amino transporter is to be understood, for example a cDNA sequence that codes an amino transporter from
Arabidopsis thaliana.
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.
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).
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.
Further, a double mutant JT16 (Tanaka & 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.
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).
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
Fox David T.
Frommer & Lawrence & Haug LLP
Hoechst Schering AgrEvo GmbH
Kallis Russell
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