We claim:
1. An isolated nucleic acid molecule comprising a sequence which encodes a protein which mediates the uptake of L-malate, succinate, and malonate.
2. An isolated nucleic acid molecule as claimed in claim 1 wherein the protein contains a PEST region, and a leucine zipper motif.
3. An isolated nucleic acid molecule as claimed in claim 1 comprising
(i) a nucleic acid sequence encoding a protein having the amino acid sequence shown in SEQ ID NO: 2 or FIG. 3;
(ii) nucleic acid sequences complementary to (i); and
(iii) a nucleic acid capable of hybridizing under stringent conditions to a nucleic acid of (i).
4. An isolated nucleic acid molecule as claimed in claim 1 comprising
(i) a nucleic acid sequence as shown in SEQ ID NO:1 or FIG. 3, wherein T can also be U;
(ii) nucleic acid sequences complementary to (i), preferably complementary to the full length nucleic acid sequence shown in SEQ ID NO: 1 or FIG. 3;
(iii) a nucleic acid capable of hybridizing under stringent conditions to a nucleic acid of (i); and
(iv) a nucleic acid molecule differing from any of the nucleic acids of (i) to (iii) in codon sequences due to the degeneracy of the genetic code.
5. An isolated nucleic acid molecule as claimed in claim 1 comprising a sequence encoding a truncation of a protein having the amino acid sequence shown in SEQ ID NO: 2 or FIG. 3; an analog, or a homolog of a protein having the amino acid sequence shown in SEQ ID NO: 2 and FIG. 3; or a truncation thereof.
6. An isolated nucleic acid molecule encoding a fusion protein comprising a protein having the amino acid sequence shown in SEQ ID NO: 2 or FIG. 3 and a heterologous protein or peptide.
7. An isolated nucleic add molecule as claimed in claim 6, wherein the heterologous protein is a selectable marker protein, or a protein involved in the metabolism of L-malate, succinate, or malonate.
8. An isolated nucleic acid molecule as claimed in claim 6, wherein the heterologous protein is a malic enzyme, a malolactic enzyme, or an enzyme involved in the metabolism of malate in plants.
9. An isolated nucleic acid molecule as claimed in claim 8, wherein the heterologous protein is a malic enzyme from S. pombe.
10. An isolated nucleic acid molecule as claimed in claim 8, wherein the heterologous protein is a malolactic enzyme from S. cerevisiae.
11. An isolated nucleic acid molecule as claimed in any one of claims 1 to 11 containing regulatory sequences for the transcription and translation of the protein.
12. A recombinant expression vector adapted for transformation of a host cell comprising a nucleic acid molecule as claimed in any one of claims 1 to 11.
13. A host cell having integrated into its genome a nucleic acid molecule as claimed in any one of claims 1 to 11.
14. A host cell as claimed in claim 13 which is a yeast strain.
15. A host cell as claimed in claim 13 which is S. cerevisiae.
16. A method for preparing a protein which mediates the uptake of L-malate, succinate, and malonate comprising (a) transferring a recombinant expression vector as claimed in claim 12 into a host cell; (b) selecting transformed host cells from untransformed host cells; (c) culturing a selected transformed host cell under conditions which allow expression of the protein; and (d) isolating the protein.
17. An isolated protein characterized in that it mediates the uptake of L-malate, succinate, and malonate.
18. An isolated protein as claimed in claim 17 further characterized in that it has part or all of the primary structural conformation and the enzymatic activity of Mae1 from S. pombe.
19. An isolated protein as claimed in claim 18 which has the amino acid sequence as shown in SEQ ID NO:2 or FIG. 3.
20. An isolated protein as claimed in claim 17, 18 or 19 which is a truncation of the protein, an analog, homolog, or isoform of the protein and truncations thereof, which has malate permease activity.
21. A fusion protein comprising a protein as claimed in claim 17, 18 or 19 and a heterologous protein or peptide.
22. A fusion protein as claimed in claim 21 wherein the heterologous protein is a malic enzyme or a malolactic enzyme.
23. Antibodies having specificity against an epitope of a protein as claimed in claim 17, 18 or 19.
24. A method of increasing uptake of L-malate, succinic acid or malonate in a cell comprising transforming the cell with a nucleic acid molecule as claimed in any one of claims 1 to 11.
25. A method of providing a microorganism with the capability of degrading malate comprising transforming the microorganism with a nucleic acid molecule as claimed in any one of claims 1 to 11.
26. A method as claimed in claim 25 wherein the microorganism is a yeast strain.
27. A method of degrading malate which comprises cultivating, in the presence of a supply of malate, a microorganism which has been transformed with a nucleic acid molecule as claimed in any one of claims 1 to 11.
28. A method of degrading malate during fermentation of wine, which method comprises, cultivating, in grape musts which contain a supply of malate, a yeast strain as claimed in claim 14 or 15.
29. A method of fermenting wine, which includes cultivating, in a wine fermentation medium which includes grape must containing a supply of malate, a yeast strain as claimed in claim 14 or 15.
30. A method for identifying a substance that mediates transport of L-malate, succinate or malonate comprising incubating a protein as claimed in claim 17, 18 or 19 with a substrate of the protein, and a test substance which is suspected of affecting the activity of the protein, and determining the effect of the substance by comparing to a control.
The claims below are in addition to those above.
All refrences to claim(s) which appear below refer to the numbering after this setence.
What is claimed is:
1. A one piece base, adjustable flex ski for use on a vehicle, said ski comprising:
a molded one piece flexible elongate ski having a front, middle and rear portion, and a top and bottom side;
said bottom side of said ski being molded substantially flat except for a center keel portion, said keel portion forming a rectangular ridge with along the length of said ski;
a first connection means at the front portion of the ski;
a second connection means on the middle top portion of the ski; and
a means of connecting said ski to said vehicle.
2. A one piece base adjustable flex ski as in claim 1 wherein said one piece flexible elongate ski further defines a one piece ski saddle having a left and right section extending upward from the middle portion on the top side of said ski.
3. A one piece base adjustable flex ski as in claim 2 wherein said second connection means comprises at least one set of holes formed by said left and right section of said ski saddle said holes being in alignment with each other.
4. A one piece base adjustable flex ski as in claim 3 further comprising a pre-load bar have a front and rear end with said front end being pivotally connected to said first connection means at the front portion of the ski, and said pre-load bar being pivotally connected at said rear end to said second connection means so as to place a pre-load on said adjustable flex ski.
5. A one piece base, adjustable flex ski as in claim 4 wherein said ski further comprises a directional control means on the bottom of said ski.
6. A one piece base adjustable flex ski as in claim 5 wherein said directional control means is at least one section of carbide runner.
7. A one piece base adjustable flex ski as in claim 5 wherein said directional control means is at least one pair of veins formed in the bottom portion of said ski.
8. A one piece base adjustable flex ski as in claim 4 wherein said ski is molded from an Ultra High Molecular Weight plastic.
9. A one piece base ski for use on a vehicle, said ski comprising:
a molded one piece flexible elongate ski having a front, middle and rear portion, and a top and bottom side;
said bottom side of said ski being molded substantially flat except for a center keel portion having right angle corners, said keel portion forming a rectangular ridge with along the length of said ski;
a first connection means at the front portion of the ski;
a second connection means on the middle top portion of the ski; and
a means of connecting said ski to said vehicle.
10. A one piece base ski as in claim 9 wherein said one piece flexible elongate ski further defines a one piece ski saddle having a left and right section extending upward from the middle portion on the top side of said ski.
11. A one piece base ski as in claim 10 wherein said second connection means comprises at least one set of holes formed by said left and right section of said ski saddle said holes being in alignment with each other.
12. A one piece base ski as in claim 11 further comprising a pre-load bar have a front and rear end with said front end being pivotally connected to said first connection means at the front portion of the ski, and said pre-load bar being pivotally connected at said rear end to said second connection means so as to place a pre-load on said adjustable flex ski and further cause a pre-load depression radius on the front portion of said ski.
13. A one piece base ski as in claim 12 wherein said ski further comprises a directional control means on the bottom of said ski.
14. A one piece base ski as in claim 13 wherein said directional control means is at least one section of carbide runner.
15. A one piece base ski as in claim 13 wherein said directional control means is at least one pair of veins formed in the bottom portion of said ski.
16. A one piece base ski as in claim 12 wherein said ski is molded from an Ultra High Molecular Weight plastic.