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1. WO1997026366 - ANTHRANILATE SYNTHASE GENE AND USE THEREOF

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[ EN ]

WHAT IS CLAIMED IS:

1. An isolated and purified DNA molecule comprising a first DNA segment encoding an anthranilate synthase substantially resistant to inhibition by free L-tryptophan or an amino acid analog of tryptophan.

2. The DNA molecule of claim 1 wherein the first DNA segment encodes a plant anthranilate synthase.

3. The DNA molecule of claim 2 wherein the first DNA segment encodes a maize anthranilate synthase.

4. The DNA molecule of claim 3 wherein the first DNA segment encodes a protein having SEQ ID NO: 16.

5. The DNA molecule of claim 3 wherein the first DNA segment consists essentially of SEQ ID NO: 12.

6. An isolated and purified DNA molecule comprising a first DNA segment encoding a maize anthranilate synthase.

7. The DNA molecule of claim 6 wherein the first DNA segment encodes a protein having SEQ ID NO:2.

8. The DNA molecule of claim 6 wherein the first DNA segment consists essentially of SEQ ID NO: 1.

9. The DNA molecule of claims 1, 2, 3, 4, 5, 6, 7 or 8 further comprising a second DNA segment encoding an amino terminal chloroplast transit peptide, which is operably linked to the first DNA segment.

10. A method of imparting tolerance to an amino acid analog of tryptophan to a plant cell comprising:
(a) introducing an expression cassette comprising a first DNA
segment encoding an exogenous anthranilate synthase
operably linked to a promoter functional in a plant cell
into cells of a susceptible plant to yield transformed plant
cells, wherein the anthranilate synthase is substantially
resistant to inhibition by free L-tryptophan or an amino
acid analog of tryptophan; and
(b) expressing the anthranilate synthase encoded by the first
DNA segment in the plant cells so as to yield an amount
of said anthranilate synthase effective to render the
transformed plant cells substantially tolerant to an amount
of free L-tryptophan or an amino acid analog of
tryptophan that inhibits the growth ofthe untransformed
cells ofthe susceptible plant.

1 1. The method according to claim 10 wherein the expression of the first
DNA segment is inducible.

12. The method according to claim 10 wherein the expression cassette further comprises plasmid DNA.

13. The method according to claim 10 wherein the first DNA segment
encoding the anthranilate synthase is introduced into plant cells by a method selected from the group consisting of electroporation,
microinjection, protoplast transformation, microprojectile bombardment, Agrobacterium-mediated transformation, and liposomal encapsulation.

14. The method according to claim 10 wherein the anthranilate synthase is a plant anthranilate synthase.

15. The method according to claim 14 wherein the plant anthranilate
synthase is a maize anthranilate synthase.

16. The method according to claim 15 wherein the maize anthranilate
synthase has SEQ ID NO: 16.

17. The method according to claim 10 wherein the first DNA segment
consists essentially of SEQ ID NO: 12.

18. The method according to claim 10 wherein the plant cells comprise cells of callus, embryos, meristematic tissue, gametic tissue, or cultured cells in suspension.

19. The method according to claim 10 wherein the amino acid analog is 5- methyltryptophan.

20. The method according to claim 10 wherein the expression cassette further comprises a second DNA segment encoding an amino terminal
chloroplast transit peptide which is operably linked to the first DNA
segment.

21. The method according to claim 10 wherein the amino acid analog is 6- methy lanthranilate .

22. The method according to claim 8 wherein the anthranilate synthase
encoded by the first DNA segment is expressed in chloroplasts of the plant cells.

23. A transformed plant regenerated from the transformed plant cells
obtained by the method of claim 10.

24. A transformed seed of the transformed plant of claim 23.

25. A method for altering the tryptophan content in a plant comprising:
(a) introducing into the cells of a susceptible plant an
expression cassette comprising a recombinant DNA
segment encoding an anthranilate synthase operably
linked to a promoter functional in a plant cell to yield
transformed plant cells, wherein the DNA segment
encodes an anthranilate synthase which is substantially
resistant to inhibition by free L-tryptophan or an amino
acid analog of tryptophan; and
(b) regenerating a differentiated plant from said transformed
plant cells wherein the cells ofthe differentiated plant
express the anthranilate synthase encoded by the DNA
segment in an amount effective to increase the tryptophan
content in the cells of the differentiated plant relative to
the tryptophan content in the cells of the untransformed
susceptible plant.

26. The method according to claim 25 wherein the tryptophan content ofthe transformed plant cells is about 1.1- to 50-fold higher than that ofthe tryptophan content ofthe susceptible plant cells of step (a).

27. A method of producing anthranilate synthase comprising:
(a) introducing an expression cassette comprising a DNA
segment encoding an anthranilate synthase into a
population of cells;
(b) expressing the DNA segment encoding the anthranilate
synthase in said cells so as to yield anthranilate synthase;
and
(c) recovering the anthranilate synthase from said cells.

28. An expression cassette comprising a first DNA segment encoding an anthranilate synthase which is substantially resistant to inhibition by free L-tryptophan or an amino acid analog of tryptophan, wherein the first
DNA segment is joined in correct reading frame at its 5 '-terminus to a second DNA segment encoding an amino-terminal chloroplast transit peptide, and wherein the first DNA segment and the second DNA
segment are under the transcriptional and translational regulatory control of regulator)' regions functional in a plant cell.

29. The expression cassette of claim 28 which further comprises a selectable marker gene or a reporter gene.

30. An isolated and purified DNA molecule comprising a DNA segment encoding transcription regulatory elements for a plant anthranilate
synthase.

31. The DNA molecule of claim 30 wherein the plant anthranilate synthase is a maize anthranilate synthase.

32. The DNA molecule of claim 30 wherein the DNA segment consists of
SEQ ID NO: 14.

33. A method of selecting transformed plant cells, comprising:
(a) introducing into a plant cell an expression cassette
comprising a first DNA segment encoding an anthranilate
synthase which is substantially resistant to inhibition by
free L-tryptophan or an amino acid analog of tryptophan
to yield a transformed plant cell; and
(b) culturing the transformed plant cell in an amount of free
L-tryptophan or an amino acid analog of tryptophan that inhibits the growth of a plant cell which does not contain
the first DNA segment.

34. An isolated and purified DNA molecule comprising a DNA segment encoding a beta subunit of an anthranilate synthase.

35. The DNA molecule of claim 34 wherein the DNA segment encodes a beta subunit of a plant anthranilate synthase.

36. The DNA molecule of claim 35 wherein the DNA segment encodes a beta subunit of a maize anthranilate synthase.

37. The DNA molecule of claim 36 wherein the DNA segment consists essentially of SEQ ID NO: 8.