Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1995-10-03
2001-10-16
Prouty, Rebecca E. (Department: 1652)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C530S395000, C536S023500
Reexamination Certificate
active
06303751
ABSTRACT:
BACKGROUND OF THE INVENTION
In the mammalian central nervous system, L-glutamate serves as a major excitatory neurotransmitter. The interaction of glutamate with its membrane bound receptors is believed to play a role in many important neuronal processes including fast synaptic transmission, synaptic plasticity and long-term potentiation. These processes are fundamental to the maintenance of life and normal human abilities such as learning and memory. Monaghan, D. T. et al., 8 Neuron 267 (1992).
Pharmacological characterization of receptors for L-glutamate has led to their classification into two families based on their biological function: the ionotropic receptors which are directly coupled to cation channels in the cell membrane, and the metabotropic receptors which function through coupling to G-proteins. The present invention concerns a member of the metabotropic family of glutamate receptors.
In addition to its role in normal human physiology, interaction of L-glutamate with its receptors is believed to play a key role in many neurological disorders such as stroke, epilepsy and head trauma, as well as neurode-generative processes such as Alzheimer's disease. Olney, R. W., 17
Drug Dev. Res
. 299 (1989). For this reason, understanding the molecular structure of human L-glutamate receptors is important for understanding these disease processes as well as for searching for effective therapeutic agents. Up to the present, the search for therapeutic agents which will bind and modulate the function of human glutamate receptors has been hampered by the unavailability of homo-geneous sources of receptors. The brain tissues commonly used by pharmacologists are derived from experimental animals (non-human) and furthermore contain mixtures of various types of glutamate receptors.
Moreover, in searching for drugs for human therapy, it is obviously desirable to use receptors which are more analogous to those in the intact human brain than are the rodent receptors employed to date. The current invention provides a human receptor which can be used to search selectively for drugs which modulate these receptors.
Recently, four metabotropic receptor subtypes (mGluR1-mGluR4) have been cloned from rat brain. Masu et al., 349 Nature 760 (1991), Houamed et al., 252 Science 1314 (1991) and Tanabe Y. et al. 8 Neuron 169 (1991). In addition, two alternately spliced versions of mGluR1 are known. Tanabe Y. et al. 8 Neuron 169 (1991).
The present invention provides a functional human metabotropic glutamate receptor to the common store of knowledge. The new receptor, called HSmGluR1, will prove especially beneficial to the development of human therapeutic agents.
SUMMARY OF THE INVENTION
The present invention provides compounds which comprise the amino acid sequence SEQ ID NO:1. In particular, the amino acid compound which is SEQ ID NO:1 is preferred.
The invention also provides nucleic acid compounds which comprise a nucleic acid sequence which encodes the amino acid compounds provided. Particularly, nucleic acid compounds which are DNA are preferred. Most preferred is the DNA compound SEQ ID NO:2. However, also preferred are those nucleic acid compounds which are sense mRNA.
Also provided by the present invention are recombinant nucleic acid vectors comprising the nucleic acids which encode SEQ ID NO:1. The preferred nucleic acid vectors are those which are DNA. Most preferred are recombinant DNA vectors which comprise the DNA sequence which is SEQ ID NO:2. A preferred DNA vector which comprises SEQ ID NO:2 is pRS117.
Moreover, recombinant DNA vectors of the present invention preferably comprise a promoter positioned to drive expression of a DNA sequence which encodes SEQ ID NO:1. Those vectors wherein said promoter functions in mammalian cells are preferred. Those mammalian vectors wherein said promoter functions in AV12 cells are preferred. The recombinant DNA expression vector most preferred is plasmid pRS121.
Restriction fragments of the preferred vectors are also provided. Particularly, the approximately 4.1 kb EcoRI and the approximately 3.8 kb BssHII/AflII restriction fragment of a vector which comprises SEQ ID NO:2 are provided.
The present invention also provides probes and primers useful for molecular biology techniques. A compound which encodes all or part of SEQ ID NO:1 or the reverse complement of a compound which encodes SEQ ID NO:1, and which is at least 18 consecutive base pairs in length is provided as a probe and/or a primer. Preferably, the 18 base pair or more compound is DNA. Most preferred for this use are the DNA compounds which are SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, or their reverse complements.
Further, this invention provides cells in which the nucleic acid compounds of the invention may be harbored. For example, oocytes wherein nucleic acid compounds of the invention express functional HSmGluR1 receptor are provided. An oocyte wherein DNA expresses functional HSmGluR1 receptor is preferred. Most preferred is an oocyte wherein sense mRNA expresses functional HSmGluR1 receptor.
Other host cells include those which are transfected with a nucleic acid compound which encodes SEQ ID NO:1. The preferred transfected host cells which encode SEQ ID NO:1 are mammalian cells and
E. Coli
. Preferred mammalian cells include AV12 cells. Preferred host cells are those which have been transfected with a recombinant DNA vector. Preferably, the DNA vector comprises SEQ ID NO:2. The most preferred transfected host cells are AV12/pRS121 and
E. coli
/pRS117.
Additionally, the invention provides a method for identifying nucleic acids homologous to a probe of the present invention, which comprises contacting the test nucleic acid with the probe under hybridizing conditions, and identifying nucleic acids which are homologous to the probe. The preferred probes for use in this method are SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.
Assays utilizing the compounds provided by the present invention are also provided. The assays provided determine whether a substance interacts with or affects the compound SEQ ID NO:1, said assays comprising contacting a functional compound of SEQ ID NO:1 with said substance, monitoring interaction by physically detectable means, and identifying those substances which effect a chosen response.
Preferably, the physically detectable means are competition with labeled glutamate, hydrolysis of phosphatidylinositol (PI), electrophysiological response in an oocyte expression system, stimulation or inhibition of adenylate cyclase or release of arachidonic acid. A most preferred glutamate competition assay utilizes radioisotopelabeled glutamate. A most preferred oocyte expression system utilizes sense mRNA.
The invention also provides a method for constructing a recombinant host cell capable of expressing a nucleic acid compound which encodes a compound which comprises SEQ ID NO:1, said method comprising transfecting a host cell with a recombinant DNA vector that comprises said nucleic acid compound. The preferred method utilizes mammalian cells as the host cells. The most preferred method utilizes AV12 cells as the mammalian host cells. A preferred method includes a DNA vector which comprises SEQ ID NO:2. A most preferred method utilizes the DNA vector pRS121.
Additionally, a method for expressing a nucleic acid sequence which encodes SEQ ID NO:1 in a recombinant host cell is provided. The method comprises culturing a transfected host cell provided by the present invention under conditions suitable for gene expression. The preferred method utilizes mammalian cells as the host cells. The most preferred method utilizes AV12 cells as the mammalian host cells. The more preferred method utilizes a recombinant DNA vector comprising SEQ ID NO:2. The most preferred method utilizes the recombinant DNA vector pRS121.
The following section provides a detailed description of the present invention. For purposes of clarity and as an aid in understanding the invention, as disclosed and claimed herein, the following items are defined below.
“mRNA”—RNA which has been transcribed eit
Burnett J. Paul
Mayne Nancy G.
Sharp Robert L.
Snyder Yvonne M.
Eli Lilly and Company
Prouty Rebecca E.
Webster Thomas D.
Wilson Alexander
LandOfFree
Human metabotropic glutamate receptor and related DNA compounds does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Human metabotropic glutamate receptor and related DNA compounds, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Human metabotropic glutamate receptor and related DNA compounds will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2614024