Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1995-05-10
2003-05-13
Kunz, Gary (Department: 1647)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S007200, C435S252300, C435S471000, C530S350000, C536S023500
Reexamination Certificate
active
06562587
ABSTRACT:
This application is a 371 of PCT/FR93/01097, filed Nov. 08, 1993.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to novel polypeptides and to the genetic material which enables them to be expressed. More specifically, it relates to novel polypeptides having an opioid receptor activity.
2. Description of the Related Art
The opioid receptors have been known for a long time as membrane receptors of the nervous system, which receptors mediate the analgesic effects of alkaloid derivatives of opium. Endogenous ligands of these receptors and their precursors have been characterized, and their role in the response to pain and stress has been widely studied (Akil et al., (1984) Annu. Rev. Neurosci. 7, 223-255). Furthermore, pharmacological studies have revealed the existence of three subtypes of opioid receptors: mu (morphine), delta (enkephalin) and kappa (dynorphin) receptors. These same studies also demonstrated that the inhibitory action of the cellular activity of these receptors was linked to the activation of G proteins (Simonds, W. F. (1988) Endocrine Rev. 9, 200-212). For this reason, these receptors are nowadays classified within the family of receptors which interact with G proteins, that is a class of receptors possessing seven transmembrane domains and encompassing approximately 80% of known receptors.
Different laboratories have attempted to clone genes encoding opioid receptors. In particular, a protein which binds opioids with a selectivity of the mu type was purified from ox brain and partially sequenced. A cDNA was then isolated using nucleotide probes derived from this partial sequence. However, the protein which is deduced from this sequence does not possess any transmembrane domain and exhibits a high degree of homology with NCAM, an adhesion molecule (Schofield et al., (1989) EMBO J. 8, 489-495). More recently, the isolation of another cDNA has been described (Xie et al., (1992) Proc. Natl. Acad. Sci. USA 89, 4124-4128), which was obtained by expression cloning. The cDNA library was constructed from human placenta, which only expressed the kappa subtype, and it was screened using a peptide derivative of dynorphin by means of an affinity enrichment technique. However, this protein possesses a relatively weak affinity for opioid ligands, does not exhibit any subtype specificity, and, finally, seems very similar to the receptor for neuromedin K. Finally, all the attempts to clone opioid receptors by PCR, which are based on homologies with receptors coupled to G proteins, have been fruitless
SUMMARY OF THE INVENTION
The present invention describes, for the first time, the cloning of genes encoding opioid receptors. The present invention also describes, for the first time, the sequence of opioid receptors and their expression in recombinant cells. The present invention thus renders it possible to obtain an improved understanding of the structure of opioid receptors and to study their mechanism of action in more detail. The present invention also renders it possible to obtain opioid receptors of very high purity and in elevated quantity, thereby permitting functional and pharmacological studies to be carried out, antibodies to be made, etc. The invention also renders it possible to prepare opioid receptor fragments of defined size, as well as all types of opioid receptor derivatives. The invention also supplies recombinant cells which express opioid receptors or opioid receptor fragments which can be used for screening ligands of these receptors (agonists, antagonists, modulators, etc.). The DNA sequences of the invention also render it possible to make probes which are capable of detecting, in biological samples, any irregularity in the expression of an opioid receptor (non-expression, mutation, polymorphism, etc.). These probes can also be used for hybridization cloning of any other cDNA encoding an opioid receptor using tissues of various origins and, in particular, of human origin, as indicated further below.
An initial subject of the invention is, therefore, a nucleotide sequence which encodes a polypeptide having an opioid receptor activity. Within the meaning of the invention, opioid receptor comprises, in particular, the delta, mu and kappa subtypes.
Preferably, the invention relates to a nucleotide sequence which encodes a polypeptide having a delta opioid receptor activity.
Still more preferably, the nucleotide sequence according to the invention is selected from among:
(a) all or part of the nucleotide sequence SEQ ID NO: 1, or of its complementary strand,
(b) any sequence which hybridizes to a sequence (a) and which encodes a polypeptide having an opioid receptor activity, and
(c) the sequences derived from sequences (a) and (b) on account of the degeneracy of the genetic code.
A quite specific embodiment of the invention is represented by a nucleotide sequence which comprises all or part of the nucleotide sequence SEQ ID NO: 1, or of its complementary strand.
The various nucleotide sequences of the invention may or may not be of artificial origin. They can be genomic, cDNA or RNA sequences, hybrid sequences, or synthetic or semi-synthetic sequences. These sequences can be obtained, for example, by screening DNA libraries (cDNA library or genomic DNA library) with probes which are developed on the basis of the sequence SEQ ID NO: 1. Such libraries can be prepared from cells of different origins using standard molecular biological techniques which are known to the person skilled in the art. The nucleotide sequences of the invention can also be prepared by chemical synthesis, in particular using the phosphoramidite method, or else using mixed methods including chemical or enzymic modification of sequences which are obtained by screening libraries.
The nucleotide sequences of the invention may be used for producing opioid polypeptides. The term opioid polypeptide denotes any polypeptide having an opioid receptor activity, and any fragment or derivative of such a polypeptide. In order to produce opioid polypeptides, that part encoding the said polypeptide is generally placed under the control of signals which enable it to be expressed in a host cell. The choice of these signals (promoters, terminators, etc.) can vary depending on the host cell employed. To this end, the nucleotide sequences of the invention can be part of a vector, which can be autonomously replicating or integrating. More specifically, autonomously replicating vectors can be prepared using sequences which ensure autonomous replication in the chosen host. As regards integrating vectors, these can be prepared, for example, using sequences which are homologous with certain regions of the genome of the host, thereby allowing integration of the vector to take place by homologous recombination. The host cells which can be used for producing opioid polypeptides of the invention by the recombinant route are eukaryotic hosts as well as prokaryotic hosts. The suitable eukaryotic hosts which may be cited are animal cells, yeasts or fungi. In particular, as regards yeasts, those of the genera Saccharomyces, Kluyveromyces, Pichia, Schwanniomyces or Hansenula may be cited. As regards animal cells, those which may be cited are COS, CHO, C127 and NIH-3T3 cells, etc. Those fungi which may be cited more specifically are Aspergillus ssp. or Trichoderma ssp. Preference is given to employing the following bacteria
E. coli
, Bacillus or Streptomyces as prokaryotic hosts.
The nucleotide sequences of the present invention can also be used in the pharmaceutical sphere, either for preparing antisense sequences which can be used within the scope of gene therapy, or, once again, for preparing probes which render it possible to detect, by hybridization experiments, expression of opioid receptors in biological samples, and to demonstrate genetic anomalies (polymorphism or mutations), or aberrant expressions.
Inhibition of the expression of certain genes by antisense sequences has turned out to be a promising strategy for controlling gene activity. Antisense sequences are sequences whos
Astra Pharma Inc.
Kunz Gary
Landsman Robert S.
Young & Thompson
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