Nucleotide sequences

Details Nucleotide sequences Nucleotide sequences

1999-10-22

2002-10-15

Kunz, Gary L. (Department: 1646)

Chemistry: molecular biology and microbiology

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

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

C435S071100, C435S071200, C435S320100, C435S325000, C435S252300, C435S254110, C530S350000, C536S023100, C536S023500, C536S024300, C536S024310

Reexamination Certificate

active

06465213

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to nucleic acid molecules encoding GABA
B
receptors, and to methods for screening for compounds that are inhibitors of transient lower esophageal sphincter relaxations (TLESR).
BACKGROUND OF THE INVENTION
GABA
B
Receptors
GABA (4-aminobutanoic acid) is an endogenous neurotransmitter in the central and peripheral nervous systems. Receptors for GABA have traditionally been divided into GABA
A
and GABA
B
receptor subtypes. GABA
B
receptors (for a review see Kerr, D. I. B. and Ong, J. (1995) Pharmac. Ther. vol. 67, pp.187-246) belong to the superfamily of G-protein coupled receptors. GABA
B
receptor agonists are useful in the treatment of central nervous system (CNS) disorders, such as for inducing muscle relaxation in spinal spasticity, cardiovascular disorders, asthma, and gut motility disorders such as irritable bowel syndrome; and as prokinetic and anti-tussive agents. GABA
B
receptor agonists have also been disclosed as useful in the treatment of emesis (WO 96/11680).
The cloning of the rat GABA
B
receptors GABA
B
R1a (SEQ ID NOs: 44 and 45) and GABA
B
R1b (SEQ ID NOs: 46 and 47) was disclosed by Kaupmann et al. ((1997) Nature, vol. 386, 239-246). The mature rat GABA
B
R1b differs from GABA
B
R1a in that the N-terminal 147 residues are replaced by 18 different residues. It is thought that the rat GABA
B
R1a and GABA
B
R1b receptor variants are derived from the same gene by alternative splicing. Cloning of the human GABA
B
R1b receptor was disclosed in WO97/46675.
Reflux
In some humans, the lower esophageal sphincter (LES) is prone to relaxing more frequently than in other humans. As a consequence, fluid from the stomach can pass into the esophagus because the mechanical barrier is temporarily lost at such times, an event hereinafter referred to as “reflux.”
Gastro-esophageal reflux disease (GERD) is the most prevalent upper gastrointestinal tract disease. Conventional therapies have sought to reduce gastric acid secretion, or reduce esophageal acid exposure by enhancing esophageal clearance, lower esophageal sphincter tone, and gastric emptying. The major mechanism behind reflux has been considered to depend on a hypotonic lower esophageal sphincter. However, recent research (e.g., Holloway & Dent (1990) Gastroenterol. Clin. N. Amer. 19, 517-535) has shown that most reflux episodes occur during transient lower esophageal sphincter relaxations (TLESR), i.e., relaxations not triggered by swallowing. It has also been shown that gastric acid secretion usually is normal in patients with GERD.
SUMMARY OF THE INVENTION
The present invention provides nucleic acid molecules encoding human and canine GABA
B
receptors. These nucleic acid molecules make it possible to screen for compounds that are agonists or antagonists of GABA
B
receptors, e.g., to identify compounds which are inhibitors of TLESR.
Consequently, the invention provides an isolated nucleic acid molecule encoding a human or canine GABA
B
receptor, or a conservative variant thereof. An “isolated nucleic acid” is a nucleic acid the structure of which is not identical to that of any naturally occurring nucleic acid or to that of any fragment of a naturally occurring genomic nucleic acid spanning more than three separate genes. The term therefor covers, for example, (a) a DNA which has the sequence of part of a naturally occurring genomic DNA molecule but is not flanked by both of the coding sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein. Specifically excluded from this definition are nucleic acids present in mixtures of (i) DNA molecules, (ii) transfected cells, and (iii) cell clones: e.g., as these occur in a DNA library such as a cDNA or genomic DNA library.
In various embodiments, the nucleic acid molecule encodes a human GABA
B
receptor 1a (SEQ ID NOs: 48 and 49), 1b (SEQ ID NOs: 50 and 51), 1c (SEQ ID NOs: 54 and 55) or 1d (SEQ ID NOs: 56 and 57); or a canine GABA
B
receptor 1a (SEQ ID NOs: 52 and 53) or 1c (SEQ ID NOs: 58 and 59). Accordingly, the invention includes the following nucleic acid molecules:
(1) a nucleic acid molecule that includes a nucleotide sequence set forth as SEQ ID NO: 48, 50, 52, 54, 56, or 58, or a degenerate variant thereof;
(2) an RNA molecule that includes a nucleotide sequence set forth as SEQ ID NO: 48, 50, 52, 54, 56, or 58, or a degenerate variant thereof, wherein T is replaced by U;
(3) a nucleic acid molecule that includes a nucleotide sequence that is capable of hybridizing under stringent conditions (e.g., is complementary) to a nucleotide sequence of (1) or (2), or to the complement of (1) or (2); and
(4) nucleic acid fragments that are at least 15 base pairs in length and which hybridize under stringent conditions to genomic DNA encoding the human or canine GABA
B
polypeptides described herein, or to the complement of such genomic DNA.
The invention also includes isolated nucleic acid molecules corresponding to genomic sequences encoding human GABA
B
receptors (SEQ ID NOs: 60 and 61), as well as nucleic acid molecules (set forth as SEQ ID NO: 70, 72, 74, 76, 78, 80, 82, and 84) encoding additional isoforms of the human GABA
B
receptor, which isoforms are generated by alternative splicing.
The nucleic acid molecules of the invention are not limited strictly to molecules including the sequences set forth as SEQ ID NOs: 48, 50, 52, 54, 56 or 58. Rather, the invention encompasses nucleic acid molecules carrying modifications such as substitutions, small deletions, insertions, or inversions, which nevertheless encode proteins having substantially the biochemical activity of the GABA
B
receptors according to the invention, and/or which can serve as hybridization probes for identifying a nucleic acid with one of the disclosed sequences. Included in the invention are nucleic acid molecules, the nucleotide sequence of which is at least 95% identical (e.g., at least 96%, 97%, 98%, or 99% identical) to the nucleotide sequence shown as SEQ ID NO: 48, 50, 52, 54, 56, or 58 in the Sequence Listing.
The determination of percent identity or homology between two sequences is accomplished using the algorithm of Karlin and Altschul (1990)
Proc. Nat'l Acad. Sci. USA
87: 2264-2268, modified as in Karlin and Altschul (1993)
Proc. Nat'l Acad. Sci. USA
90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990)
J. Mol. Biol
. 215:403-410. BLAST nucleotide searches are performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches are performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST is utilized as described in Altschul et al. (1997)
Nucleic Acids Res
. 25: 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) are used. See http://www.ncbi.nlm.nih.gov.
The term “stringent hybridization conditions” is known in the art from standard protocols (e.g., Current Protocols in Molecular Biology, editors F. Ausubel et al., John Wiley and Sons, Inc. 1994) and is to be understood as conditions as stringent as those defined by the following: hybridization to filter-bound DNA in 0.5 M NaHPO
4
(pH 7.2), 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at +65° C., and washing in 0.1×SSC/0.1% SDS at +6

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