Vitamin B metabolism proteins
OwnerRebecca E. Cahoon, Saverio Carl Falco +1inventors
InventorsRebecca E. Cahoon, Saverio Carl Falco, Karlene H. Butler
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Abstract
This invention relates to an isolated nucleic acid fragment encoding a thiamin pyrophosphokinase. The invention also relates to the construction of a chimeric gene encoding all or a portion of the thiamin pyrophosphokinase, in sense or antisense orientation, wherein expression of the chimeric gene results in production of altered levels of the thiamin pyrophosphokinase in a transformed host cell.
Claims12
An isolated nucleic acid fragment encoding a thiamin pyrophosphokinase comprising a member selected from the group consisting of:
- (a) an isolated nucleic acid fragment encoding an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in a member selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:8;
- (b) an isolated nucleic acid fragment that is complementary to (a).
A thiamin pyrophosphokinase polypeptide comprising all or a substantial portion of the amino acid sequence set forth in a member selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:8.
A method of obtaining a nucleic acid fragment encoding a substantial portion of an amino acid sequence encoding a thiamin pyrophosphokinase comprising:
- (a) synthesizing an oligonucleotide primer corresponding to a portion of the sequence set forth in any of SEQ ID NOs:1, 3, 5 and 7;
- and
- (b) amplifying a cDNA insert present in a cloning vector using the oligonucleotide primer of step (a) and a primer representing sequences of the cloning vector
- wherein the amplified nucleic acid fragment encodes a substantial portion of an amino acid sequence encoding a thiamin pyrophosphokinase.
A method for evaluating at least one compound for its ability to inhibit the activity of a thiamin pyrophosphokinase, the method comprising the steps of:
- (a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a thiamin pyrophosphokinase, operably linked to suitable regulatory sequences;
- (b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of the thiamin pyrophosphokinase encoded by the operably linked nucleic acid fragment in the transformed host cell;
- (c) optionally purifying the thiamin pyrophosphokinase expressed by the transformed host cell;
- (d) treating the thiamin pyrophosphokinase with a compound to be tested;
- and
- (e) comparing the activity of the thiamin pyrophosphokinase that has been treated with a test compound to the activity of an untreated thiamin pyrophosphokinase,
- thereby selecting compounds with potential for inhibitory activity.
Dependencies are inferred from the claim text.