Galanin receptors, nucleic acids, transformed cells and uses...

Details Galanin receptors, nucleic acids, transformed cells and uses... Galanin receptors, nucleic acids, transformed cells and uses...

1996-08-15

2002-09-10

Duffy, Patricia A. (Department: 1645)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S007210, C435S069100, C435S070100, C435S320100, C435S325000, C435S361000, C530S350000, C536S023500

Reexamination Certificate

active

06447996

ABSTRACT:

The present invention relates to new polypeptides and to the genetic material permitting their expression. More especially, it relates to new polypeptides having galanin receptor activity.
Galanin is a ubiquitous neuropeptide of 29 amino acids in mammals (30 in man) which controls miscellaneous biological functions, namely (i) endocrine secretions (insulin, somatostatin, glucagon, growth hormone, etc.), (ii) muscle tone in the digestive tract, (iii) control of behaviour (food intake, perception of painful stimuli, learning, memory, pain, etc.), through a neuromodulatory effect at central nervous system level. This non-exhaustive list of the effects of galanin, most often demonstrated in animals, explains the growing interest of pharmacologists in this neuropeptide. Selective molecules (galanin agonist or antagonist) would constitute potential pharmacological agents in endocrinology, neurology and psychiatry (Bartfai et al., (1992) TIPS 13, 312-317).
A study of the mechanism of action of galanin shows that it acts via specific membrane receptors. The biochemical and molecular characterization of the receptor (Chen et al., (1993) PNAS 90, 3845-3849, Fisone et al., (1989) Eur. J. Biochem. 180, 269-276) indicates that it belongs to the family of receptors coupled to G proteins. Depending on the target tissues, the peptide inhibits adenylate cyclase, decreases intracellular calcium, blocks voltage-dependent calcium channels or activates ATP-sensitive potassium channels. Studies of structure-activity relationships, by means of C- and N-terminal fragments of galanin and of chimeric peptides, have shown that (1) irrespective of the tissue, no partial sequence of the peptide is sufficient for obtaining full activity, (2) depending on the tissues, the first two N-terminal amino acids and the C-terminal domain 16-29 of galanin are not always essential for its activity. These observations suggest the existence of subtypes of galaninergic receptors.
The present invention describes for the first time the cloning of a gene coding for a human galaninergic receptor. The present invention also describes for the first time the sequence of galaninergic receptors and their expression in recombinant cells. The present invention thus permits a better understanding of the structure of the galaninergic receptors and enables their mechanism of action to be studied more meticulously. The present invention also enables galaninergic receptors of very high purity to be obtained in large amounts, enabling functional and pharmacological studies, the production of antibodies, and the like, to be carried out. The invention also makes it possible to prepare fragments of galaninergic receptors of defined size, as well as all kinds of derivatives of galaninergic receptors. The invention also provides recombinant cells expressing galaninergic receptors or fragments of galaninergic receptors, which are usable for the screening of ligands for these receptors (agonists, antagonists, modulators, and the like). The DNA sequences of the invention also make it possible to produce probes capable of detecting any irregularity in the expression of a galaninergic receptor (non-expression, mutation, polymorphism, and the like) in biological samples. These probes can also be used for the cloning by hybridization of any other cDNA coding for a galaninergic receptor, from tissues of diverse origins, as indicated later.
Hence a first subject of the invention lies in a nucleotide sequence coding for a polypeptide having galaninergic receptor activity. For the purposes of the invention, galaninergic receptor comprises, in particular, all the potential subtypes.
More preferably, the nucleotide sequence according to the invention is chosen from:
(a) all or part of the nucleotide sequence SEQ ID No. 1 or of its complementary strand,
(b) any sequence hybridizing with a sequence (a) and coding for a polypeptide having galaninergic receptor activity, and
(c) the sequences derived from the sequences (a) and (b) as a result of the degeneracy of the genetic code.
A very special embodiment of the invention is represented by a nucleotide sequence comprising all or part of the nucleotide sequence SEQ ID No. 1 or of its complementary strand.
The different nucleotide sequences of the invention may be of artificial origin or otherwise. They can be genomic, cDNA or RNA sequences, hybrid sequences or synthetic or semi-synthetic sequences. These sequences may be obtained, for example, by the screening of DNA libraries (cDNA library, genomic DNA library) by means of probes produced on the basis of the sequence SEQ ID No. 1. Such libraries may be prepared from cells of different origins by standard techniques of molecular biology known to a person skilled in the art. The nucleotide sequences of the invention may also be prepared by chemical synthesis, in particular according to the phosphoramidite method, or alternatively by mixed methods including the chemical or enzymatic modification of sequences obtained by the screening of libraries.
The nucleotide sequences of the invention may be used for the production of galaninergic polypeptides. The term galaninergic polypeptide denotes any polypeptide having galaninergic receptor activity, and any fragment or derivative of such a polypeptide. For the production of galaninergic polypeptides, the portion coding for the said polypeptide is generally placed under the control of signals permitting its expression in a cell host. The choice of these signals (promoters, terminators, and the like) may vary in accordance with the cell host used. To this end, the nucleotide sequences of the invention may form part of a vector, which can be autonomously replicating or integrative. More especially, autonomously replicating vectors may be prepared using sequences which are autonomously replicating in the chosen host. As regards integrative vectors, these may be prepared, for example, using sequences which are homologous to certain regions of the host's genome, permitting integration of the vector by homologous recombination. The cell hosts which can be used for the production of the galaninergic polypeptides of the invention by methods employing recombination are either eukaryotic or prokaryotic hosts. Among suitable eukaryotic hosts, animal cells, yeasts or fungi may be mentioned. In particular, as regards yeasts, yeasts of the genus Saccharomyces, Kluyveromyces, Pichia, Schwanniomyces or Hansenula may be mentioned. As regards animal cells, COS, CHO, C127, NIH-3T3, and the like, cells may be mentioned. Among fungi, Aspergillus ssp. or Trichoderma ssp. may be mentioned more especially. As prokaryotic hosts, it is preferable to use the following bacteria:
E.coli,
Bacillus or Streptomyces.
The nucleotide sequences of the present invention can also be used in the pharmaceutical field, either for the production of antisense sequences which can be used in the context of a gene therapy, or for the production of probes permitting the detection, by hybridization experiments, of the expression of galaninergic receptors in biological samples and the demonstration of genetic abnormalities (polymorphism, mutations) or of aberrant expressions.
The inhibition of the expression of certain genes by antisense sequences has proved to be a promising strategy in the control of the activity of a gene. Antisense sequences are sequences whose transcription product is complementary to the coding strand of a given gene and is, as a result, capable of hybridizing specifically with the transcribed mRNA or with the gene, inhibiting its transcription or its translation into protein. Thus, a subject of the invention is the antisense sequences capable of at least partially inhibiting the production of galaninergic polypeptides as are defined above. Such sequences can consist of all or part of the nucleotide sequences defined above. They are generally sequences or fragments of sequences complementary to sequences coding for peptides of the invention. Such sequences may be obtained from the sequence SEQ ID No. 1, by fragmentation, and the like,

Affiliated with

Also associated with

Rating

Galanin receptors, nucleic acids, transformed cells and uses... does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Galanin receptors, nucleic acids, transformed cells and uses..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Galanin receptors, nucleic acids, transformed cells and uses... will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2877425