Nucleic sequences coding for an AT2 interacting proteins interacting with the AT2 receptor and their applications

• US 6,835,539 B1
• Filed: 10/08/2001
• Issued: 12/28/2004
• Est. Priority Date: 08/04/1998
• Status: Expired due to Fees

• Current Assignee: Arthur Donny Strosberg, Clara Nahmias, Nathalie Elbaz
• Original Assignee: Arthur Donny Strosberg, Clara Nahmias, Nathalie Elbaz
• Inventors: Strosberg, Arthur Donny, Nahmias, Clara, Elbaz, Nathalie
• Primary Examiner(s): Wax, Robert A.
• Application Number:
  US09/762,194
• Time in Patent Office: 1,177 Days
• Field of Search: 435/252.33, 435/254.2, 435/320.1, 435/358, 435/6, 536/23.5
• US Class Current: 435/6.18
• CPC Class Codes: C07K 14/47 from mammals
• CPC Class Codes: C07K 2319/00 Fusion polypeptide

Abstract

The invention concerns nucleic sequences coding for a protein capable of interacting with the AT2 receptor, oligonucleotides included in said sequences, their applications as probes and for expressing said proteins, vectors useful for said expression, host cells containing said vectors, and study model of AT2 receptor. The invention also concerns said proteins and their uses. Said isolated nucleic acid fragment coding for a protein capable of binding with the AT2 receptor is selected among the group consisting of the sequences SEQ ID NO: 1, 3, 5, 7 and 9.

Claims

1. Isolated nucleic acid fragment, encoding a protein capable of binding to the AT2 receptor, which fragment is selected from the group consisting of the sequences SEQ ID NO:

1, 3, 7 and 9.

4. Transcripts, characterized in that they are complementary to the sequences according to claim 1.

5. Recombinant, cloning and/or expression vector, characterized in that it comprises a nucleotide sequence according to claim 1 or sequence SEQ ID NO:

5.

6. Transformed host cell, characterized in that it comprises a vector according to claim 5.

2. Probe or primer, wherein it comprises from 20 to 400 bp of SEQ ID NO:

1, 3, 5, 7 or 9, or to the complementary sequences of said sequences.

3. Fragment according to claim 2, characterized in that it is selected from the group consisting of the sequences SEQ ID NO:

5, SEQ ID NO;

10, SEQ ID NO;

11 and SEQ ID NO;

12.

11. Method of detection of nucleic acid molecule encoding a protein of the ATIP family, comprising:

extracting total RNA from a biological sample,

obtaining the corresponding cDNA,

amplifying said cDNA with a pair of primers as defined in claim 2,

hybridizing said amplified cDNA with a probe as defined in claim 2 under the following hybridization conditions;

prehybridization and hybridization in 45% formamide, 9% dextran sulphate, 0.2% BSA, 0.2% polyvinylpyrrolidone, 0.2% Ficoll, 0.1% sodium pyrophosphate, 0.01% SDS, 0.05 mM Tris pH 7.5, 0.9 M NaCl and rinses to stringency;

1×

SSC, 0.1% SDS.

7. Transformed host cells, characterized in that they consist of a suitable yeast strain cotransformed with at least two vectors which respectively encode (i) a so-called bait protein selected from the group consisting of a fragment containing at least SEQ ID NO:

5 encoding a fragment of the AT2 interacting protein, and a fragment encoding at least the C-terminal end of the AT2 receptor corresponding to cytoplasmic domain of said AT2 receptor, which bait protein is fused with a protein selected from the group consisting of the DNA-binding domain of a transcription factor and the activation domain of the same transcription factor and (ii) a so-called prey protein, selected from the group consisting of a fragment containing at least SEQ ID NO;

5 encoding a fragment of the AT2 interacting protein, a fragment encoding at least said C-terminal end of the AT2 receptor and any other polypeptide corresponding to a sequence contained in a cDNA library, which prey protein is fused with a protein selected from the group consisting of the DNA-binding domain of a transcription factor and the activation domain of the same transcription factor, which vectors comprise, in addition, selectable markers.

8. Transformed host cell according to claim 7, characterized in that it consists of a suitable yeast strain cotransformed with three vectors which respectively encode (i) a bait corresponding to a fragment encoding the C-terminal end of the AT2 receptor corresponding to cytoplasmic domain of said AT2 receptor fused with a protein selected from the group consisting of the DNA-binding domain of a transcription factor and the activation domain of the said transcription factor, (ii) a fragment containing at least SEQ ID NO:

5 encoding a fragment of the AT2 interacting protein, fused with a protein selected from the group consisting of the DNA-binding domain of a transcription factor and the activation domain of the said transcription factor and (iii) a polypeptide corresponding to a sequence contained in a cDNA library, which vectors comprise, in addition, selectable markers.

9. Transformed host cell according to claim 7, characterized in that it consists of a suitable yeast strain cotransformed with two vectors which respectively encode (i) a fragment containing at least the sequence SEQ ID NO:

5 encoding a fragment of the AT2 interacting protein, fused with a protein selected from the group consisting of the DNA-binding domain of a transcription factor and the activation domain of the said transcription factor and (ii) a polypeptide corresponding to a sequence contained in a cDNA library, fused with a protein selected from the group consisting of the DNA-binding domain of a transcription factor and the activation domain of the said transcription factor, which vectors comprise, in addition, selectable markers.

10. Transformed host cell according to claim 7, characterized in that it consists of a suitable yeast strain cotransformed with two vectors, namely (i) a vector encoding a fragment containing at least the SEQ ID NO:

5 encoding a fragment of the AT2 interacting protein, mutated or not, fused with a protein selected from the group consisting of the DNA-binding domain of a transcription factor and the activation domain of the said transcription factor and (ii) a vector encoding a fragment containing the C-terminal end of the AT2 receptor corresponding to positions 314-363 of said AT2 receptor, mutated or not, fused with a protein selected from the group consisting of the DNA-binding domain of a transcription factor and the activation domain of the said transcription factor, which vectors comprise, in addition, selectable markers, one of the two vectors necessarily encoding a mutated protein.