# US 7,465,568 B2 · Essential fungal polynucleotides, polypeptides, and methods of use

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- Number: US 7,465,568 B2
- Document ID: US7465568B2
- Status: Granted
- Kind: B2
- Application number: 10/424,324
- Filed: 2003-04-25
- Published: 2007-01-18
- Granted: 2008-12-16
- Earliest claimed: 2002-04-26
- Assignees: Bristol-Myers Squibb Company
- Inventors: Ying-Kai Wang; Mengping Liu; Brian A. Dougherty; Matthew D. Healy; Daniel B. Davison; Charles E. Mazzucco; Trina C. Maurice
- Web page: https://patentgrep.com/patent/US7465568B2

## CPC

- C07K 14/395: from Saccharomyces
- C07K 14/38: from Aspergillus
- C07K 14/40: from Candida
- C07K 2299/00: Coordinates from 3D structures of peptides, e.g. proteins or enzymes
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

The presnt invention provides essential fungal polynucleotides and their encoded polypeptides, homologues thereof and their uses. Additionally, the invention provides methods for the identification of essential polynucleotides and fungal strains which may be used for drug screening.

## Claims

### Claim 1

An isolated nucleic acid molecule consisting of a polynucleotide sequence selected from the group consisting of:
(a) an isolated polynucleotide encoding a polypeptide consisting of amino acids 1 to 346 of SEQ ID NO:12; and
(b) an isolated polynucleotide encoding a polypeptide consisting of amino acids 2 to 346 of SEQ ID NO:12;
wherein said encoded polypeptide has keto-steroid reductase activity.

### Claim 2 (depends on 1)

The isolated nucleic acid molecule of claim 1, wherein said polynucleotide is (a).

### Claim 3 (depends on 2)

The isolated nucleic acid molecule of claim 2, wherein said polynucleotide consist of nucleotides 1 to 1038 of SEQ ID NO:1.

### Claim 4 (depends on 1)

The isolated nucleic acid molecule of claim 1, wherein said polynucleotide is (b).

### Claim 5 (depends on 4)

The isolated nucleic acid molecule of claim 4, wherein said polynucleotide consist of nucleotides 4 to 1038 of SEQ ID NO:1.

### Claim 6 (depends on 1)

A recombinant vector consisting of the isolated nucleic acid molecule of claim 1 operably linked to a promoter at the 5′ end and operably linked to a stop codon at the 3′ end, wherein said vector comprises an origin of replication and a selectable marker.

### Claim 7 (depends on 6)

An isolated recombinant host cell comprising the vector sequence of claim 6.

### Claim 8 (depends on 7)

A method of making an isolated polypeptide comprising:
(a) culturing the isolated recombinant host cell of claim 7 under conditions such that said polypeptide is expressed; and
(b) recovering said polypeptide.

### Claim 9 (depends on 1)

An isolated nucleic acid molecule consisting of a polynucleotide having a nucleotide sequence that is at least 95.0% identical to a polynucleotide sequence provided in claim 1, wherein percent identity is calculated using a CLUSTALW global sequence alignment using default parameters, and wherein said polynucleotide encodes a polypeptide that has keto-steroid reductase activity.

### Claim 10

An isolated nucleic acid molecule consisting of a polynucleotide encoding a polypeptide that is at least 95.0% identical to amino acids 2 to 346 of SEQ ID NO:12, wherein percent identity is calculated using a CLUSTALW global sequence alignment using default parameters, and wherein said polynucleotide encodes a polypeptide that has keto-steroid reductase activity.

### Claim 11

An isolated polynucleotide encoding a polypeptide consisting of at least 318 contiguous amino acids of SEQ ID NO:12, wherein said polypeptide has keto-steroid reductase activity.

### Claim 12 (depends on 11)

The isolated nucleic acid molecule of claim 11, wherein said polynucleotide consists of at least 954 contiguous nucleotides of SEQ ID NO:1.

### Claim 13 (depends on 1)

An isolated polynucleotide consisting of the complete complementary sequence of (a) or (b) of claim 1.