Abstract The cloning, nucleotide and deduced amino acid sequences of the gene encoding endothelial nitric oxide synthase are described. The sequence contains binding domains for calcium/calmodulin, FMN, FAD and NADPH. The enzyme has a molecular weight of 133,413 M r . The amino terminal portion of the enzyme exhibits a proline-rich region and several sites for proline-directed phosphorylation as well as a potential substrate site for acyl transferase. DNA probes prepared from the nucleic acid sequence are useful in research and diagnostically to determine the level of nitric oxide synthase mRNA expressed by endothelial cells both in cell culture and in intact tissues. These probes are also useful for detecting genetic abnormalities. The nitric oxide synthase gene is transfected into blood vessels in vivo for enhanced synthesis of nitric oxide synthase, resulting in increased production of nitric oxide. The gene is also transfected into host cells that do not normally express the enzyme for production of endothelial nitric oxide synthase in large volumes.
An isolated nucleic acid molecule having a sequence encoding bo vine endothelial nitric oxide synthase wherein the sequence encodes a protein having the amino acid sequence set forth in SEQ ID NO: 2.
Claim 2
Dependent Claim
33 words
An isolated nucleic acid molecule having a sequence encoding bo vine endothelial nitric oxide synthase wherein the sequence encodes a protein having the amino acid sequence set forth in SEQ ID NO: 2.
Claim 3
Dependent Claim
15 words
The molecule of claim 1 having the nucleic acid sequence of SEQ ID NO: 1.
Claim 4
Dependent Claim
11 words
The molecule of claim 1 further comprising a label for detection.
Claim 5
Dependent Claim
19 words
A vector comprising the molecule of claim 1 wherein said vector is suitable for expression in a host cell.
Claim 6
Dependent Claim
20 words
A host cell comprising the vector of claim 4 selected from the group consisting of procaryotic, yeast, and mammalian cells.
Claim 7
Dependent Claim
14 words
The host cell of claim 5 wherein said host cell is an endothelial cell.
Claim 8
Dependent Claim
68 words
An endothelial nitric oxide synthase probe comprising a nucleic acid having the sequence set forth in SEQ ID NO: 1 or a fragment thereof selected from the group or fragments consisting of nucleotides 30 to 3645 of SEQUENCE LISTING ID NO. 1, nucleotides 700 to 2900 of SEQUENCE LISTING ID NO. 1, and the nucleic acids corresponding to amino acids 1 to 120 of SEQ ID NO: 2.
Claim 9
Dependent Claim
68 words
An endothelial nitric oxide synthase probe comprising a nucleic acid having the sequence set forth in SEQ ID NO: 1 or a fragment thereof selected from the group or fragments consisting of nucleotides 30 to 3645 of SEQUENCE LISTING ID NO. 1, nucleotides 700 to 2900 of SEQUENCE LISTING ID NO. 1, and the nucleic acids corresponding to amino acids 1 to 120 of SEQ ID NO: 2.
Claim 10
Dependent Claim
15 words
The probe of claim 7 wherein the nucleic acid is labelled with a detectable marker.