NPY family member

Details NPY family member NPY family member

2000-09-01

2003-06-17

Kunz, Gary (Department: 1697)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S320100, C435S325000, C514S002600, C514S012200, C514S014800, C530S300000, C530S326000, C536S023100

Reexamination Certificate

active

06579700

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to isolated polynucleotides encoding PY peptides. The present invention also relates to isolated PY peptides and functionally equivalent fragments thereof, and to methods of treatment involving administration of these peptides.
BACKGROUND OF THE INVENTION
The neuropeptide Y (NPY) gene family, which also includes peptide YY (PYR) and pancreatic polypeptide (PP), is an example of multiple gene duplication events giving rise to a range of structurally related but functionally distinct gene products (1). NPY is one of the most highly conserved peptides known (for example, with only three amino acid differences between human and shark NPY), suggesting that this peptide subserves evolutionary old and important functions. In the mammalian nervous system, NPY is one of the most abundant neuropeptides and acts both centrally and peripherally to regulate the cardiovascular system. It also modulates a wide range of other important physiological activities, including appetite, central endocrine secretion, anxiety, and reproduction (2). On the other hand, PYY is secreted from endocrine cells in the lower small intestine, colon, and pancreas. It acts in an inhibitory nature on the gastrointestinal tract, including inhibition of gastric acid secretion, gastric emptying, pancreatic exocrine secretion, and gut motility (3). The third member of the NPY family, PP, is secreted by cells within the endocrine and exocrine pancreas and specifically inhibits the section of enzymes and bicarbonate from the exocrine pancreas (4).
Analysis of the structure and localization of the genes encoding NPY, PYY and PP has suggested that these genes arose from an initial gene duplication event that generated the NPY and PYY genes, followed by a subsequent duplication of the PYY gene to create the PP gene. The human NPY gene has been mapped to chromosome 7, while the PYY and PP genes lie only 10 kb apart on chromosome 17q21.1 (5). Consistent with this evolution by gene duplication, all three genes share a common intron/exon structure, although the three introns of the NPY gene (in all species studied to date) are much larger than the corresponding introns in the PYY and PP genes. The overall nucleotide sequence similarity between the three members of the gene family is restricted to the coding regions and ranges from approximately 55% (NPY vs. PYY) to 38% (NPY vs. PP) and 30% (PY vs. PP).
SUMMARY OF THE INVENTION
The present inventors have now identified a novel member of the NPY gene family. This gene, designated PY, appears to be expressed in a range of tissue types including lung, kidney, liver, ovary, adrenal gland, heart, pancreas, testis and brain. This pattern of expression is consistent with other members of the NPY gene family and suggests that the PY peptide plays an important role in central and peripheral nervous and endocrine systems.
Accordingly, in a first aspect the present invention provides an isolated polynucleotide encoding a PY peptide or a functionally equivalent fragment thereof.
The term “isolated polynucleotide” is intended to refer to one or both of the following: a polynucleotide not immediately contiguous with both of the coding sequences with which it is immediately contiguous (i.e., one at the 5′ end and one at the 3′ end) in the naturally-occurring genome of the organism from which the polynucleotide is derived; or a polynucleotide which is substantially free of a nucleic acid with which it occurs in the organism from which the polynucleotide is derived.
In one preferred embodiment the isolated polynucleotide encodes a human pre-pro PY peptide of about 33 amino acids or a mature amidated peptide of about 14 amino acids or a non-amidated peptide of 15 amino acids.
In another preferred embodiment the isolated polynucleotide encodes a baboon pre-pro PY peptide of about 63 amino acids or a mature peptide of about 35 amino acids.
More preferably, the isolated polynucleotide encodes a human PY peptide including an amino acid sequence substantially corresponding to amino acids 19 to 33 in SEQ ID NO:2 or a baboon PY peptide having a sequence substantially corresponding to amino acids 29 to 63 in SEQ ID NO;4.
In a further preferred aspect, the polynucleotide includes
(a) a sequence of nucleotides as shown in SEQ ID NO:1 or SEQ ID NO:3 or a functional equivalent thereof;
(b) a sequence complementary to the sequence of paragraph (a); or
(c) a sequence which selectively hybridises to a sequence in paragraph (a) or paragraph (b).
In a preferred embodiment, the sequence defined in paragraph (c) hybridises to a sequence in paragraph (a) or paragraph (b) under stringent conditions.
When used herein, “high stringency” refers to conditions that (i) employ low ionic strength and high temperature for washing after hybridization, for example, 0.1×SSC and 0.1% (w/v) SDS at 50° C.; (ii) employ during hybridization conditions such that the hybridization temperature is, 25° C. lower than the duplex melting temperature of the hybridizing polynucleotides, for example 1.5×SSPE, 10% (w/v) polyethylene glycol 6000, 7% (w/v) SDS, 0.25 mg/ml fragmented herring sperm DNA at 65° C.; or (iii) for example, 0.5M sodium phosphate, pH 7.2, 5 mM EDTA, 7% (w/v) SDS (28) and 0.5% (w/v) BLOTTO at 70° C.; or (iv) employ during hybridizational denaturing agent such as formamide, for example, 50% (v/v) formamide with 5×SSC, 50 mM sodium phosphate (pH 6.5) and 5×Denhardt's solution at 42° C.; or (v) employ, for example, 50% (v/v) formamide, 5×SSC, 50 mM sodium phosphate (pH 6.8), 0.1% (w/v) sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 &mgr;g/ml) and 10%. dextran sulphate at 42° C.
In a preferred embodiment, the polynucleotide is less than 5000 nucleotides in length, more preferably less than 2000 nucleotides in length, more preferably less than 1000 nucleotides in length and more preferably less than 500 nucleotides in length.
In a preferred embodiment the polynucleotide defined in paragraph (a) includes a sequence characterised by nucleotides 150 to 194 of SEQ ID NO:1 or nucleotides 580 to 684 of SEQ ID NO:3 or a functional equivalent of either of these sequences.
By “functional equivalent” we mean a sequence which differs from the sequence defined in SEQ ID NO:1 or SEQ ID NO:3 but which, through the degeneracy of the genetic code, encodes a polypeptide substantially as shown in SEQ ID NO:2 or SEQ ID NO:4.
In a further preferred embodiment, the polynucleotides of the present invention share at least 50% homology, more preferably at least 70% homology, more preferably at least 80% homology, more preferably at least 90% homology and more preferably at least 95% homology with a sequence shown in SEQ ID NO:1 or SEQ ID NO:3 wherein the homology is calculated by the BLAST program blastn as described in Altschul. S. F., Madden, T. L. Schaffer. A. A. Zhang. J., Zhang, Z., Miller, W. And Lipman. D. J. (1997) “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs”, Nucleic Acids Research 25(171:3389-3402.
The polynucleotide of the present invention may comprise DNA or RNA sequences.
The isolated polynucleotide may be incorporated into plasmids or expression vectors (including viral vectors), which may then be introduced into suitable bacterial, yeast, insect and mammalian host cells. Such host cells may be used to express the PY peptide encoded by the isolated polynucleotide molecule.
Accordingly, in a second aspect, the present invention provides a mammalian, insect, yeast or bacterial host cell transformed with the polynucleotide of the first aspect.
In a third aspect, the present invention provides a method of producing PY peptides or functionally equivalent fragments thereof, comprising culturing the host cell of the second aspect under conditions enabling the expression of the polynucleotide molecule and optionally recovering the PY peptide or fragments thereof.
The terms peptides, proteins, and polypeptides are used interchangeably herein.
Preferably, the host cell

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