Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-11-05
2002-05-07
Carlson, Karen Cochrane (Department: 1653)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C536S022100, C530S350000, C435S071100
Reexamination Certificate
active
06384204
ABSTRACT:
FIELD OF INVENTION
This invention relates to a novel DNA sequence, and derivatives thereof, useful in the screening of compounds that are agonistic or antagonistic to seronergic receptor activity.
BACKGROUND
The enormous phenotypic diversity of neural cell types implies a corresponding complexity of gene-specific transcription factor combinations required to regulate thousands of genes in the appropriate stage and cell type-specific manner (He, X., and Rosenfeld, M. G., “Mechanisms of complex transcriptional regulation: implications for brain development”
Neuron
7:183-196, 1991; Mandel, G., and McKinnon, D, “Molecular basis of neural-specific gene expression”
Annu. Rev. Neurosci.
16:323-345, 1993; Struhl, K., “Mechanisms for diversity in gene expression patterns”
Neuron
7:177-181, 1991). Indeed, several members of different transcription factor classes, such as homeodomain, zinc-finger and basic helix-loop-helix proteins, function to regulate neural cell-type identity in specific regions of both the vertebrate and invertebrate nervous systems. Many of these genes are expressed early in neural development, which suggests they play a critical role in neurogenesis and neuronal patterning (Tanabe, Y., and Jessell, T. M., “Diversity and pattern in the developing spinal cord”
Science
274:1115-1124, 1996). However, an understanding of the functional interplay between different transcription factors and the neural genes they regulate is just beginning to emerge. Little ia known about the identity and specific functions of transcription factors which operate particular differentiation programs involved in the appearance and maintenance of specific neural cell phenotypes.
The central serotonin (5-HT) neurotransmitter system consists of a relatively small population of morphologically diverse neurons whose cell bodies are present largely within the limits of the midbrain/hindbrain raphe nuclei and particular regions of the reticular formation (Steinbusch, H. W. M.,
Neuroscience
6:557-618, 1981). Although there are only about 20,000 serotonergic neurons in the rat brain the extensive axonal projection system arising from these neurons bears a tremendous number of collateral branches so that the 5-HT system densely innervates nearly all regions of the central nervous system (Halliday, G., Harding, A., and Paxinos, G. (1995) in
The rat nervous system
(Paxinos, G. ed), 2nd Ed., pp. 929-974, Academic Press, San Diego; Jacobs, B. L., and Azmitia, E. C.,
Physiological Reviews
72:165-220, 1992). Given its widespread distribution it is not surprising that 5-HT has been implicated in the control of numerous neural systems which mediate such functions as cognition, aggression, and perception (Heninger, G. R.,
Proc. Natl. Acad. Sci. USA
94:4823-4824, 1997). Abnormal function of the central 5-HT system has been implicated in several psychiatric maladies such as depression, anxiety, and eating disorders. The people afflicted by diseases caused or potentiated by abnormalities in the central serotonin 5-HT neurotransmitter system numbers in the millions. In this regard, this system is the target of several highly effective pharmacological agents that are used widely to treat these conditions. However, the current pharmacological agents may posses cross reactivity with other components of the central nervous system resulting in unwanted or debilitating side effects. Additionally, despite the clear importance of the central serotonin 5-HT system in a wide range of CNS processes and clinical disorders little is known about the genetic mechanisms which control the specification and differentiation of serotonergic neurons.
What is needed are reagents and methods that can be utilized in the screening of pharmacological compounds that can be utilized in the treatment of neurological diseases involving the central serotonin (5-HT) neurotransmitter system.
SUMMARY OF THE INVENTION
The present invention generally relates to compositions and methods of identifying and testing seronegic receptor agonists and antagonists. In addition, the invention relates to methods to identify other members of the EST transcription factor family, methods to identify homologs of Pet-1 which are native to other tissue or cell types or specific transcription factors for other neuronal cell types and methods to generate reagents derived from the invention.
The present invention contemplates employing a gene sequence (SEQ ID NO:1) that encodes a transcription factor specific for central serotonin 5-HT neurons. In one embodiment, the present invention contemplates a composition comprising isolated and purified DNA having an oligonucleotide sequence of: Pet-1 cDNA having the nucleotide sequence of SEQ ID NO:1. Such DNA may readily be inserted into expression constructs and the present invention contemplates such constructs as well as their use. The present invention also contemplates RNA transcribed from the above-indicated cDNA as well as protein (typically purified protein) translated from this RNA. Moreover, the present invention contemplates antibodies produced from immunizing with this translated protein.
The present invention also contemplates transgenic animals comprising the above-indicated DNA (i.e. the “transgene”) or portions thereof. In a particular embodiment, the transgenic animal of the present invention may be generated with the transgene contained in an inducible, tissue specific promotor.
The present invention also contemplates using the above-named compositions in screening assays. The present invention is not limited by the particular method of screening. In one embodiment cells are used such as, but not limited to, transformed cell lines. In another embodiment primary cells may be used. The present invention is not limited to the nature of the transfection construct. The transfection constructs utilized will be the optimal constructs available for the cell line chosen at the time of setting up the assay. In one embodiment, the present invention contemplates screening suspected compounds in a system utilizing transfected cell lines. In one embodiment, the cells may be transfected transiently. In another embodiment, the cells may be stably transfected. In yet another embodiment translation products of the invention may be used in a cell-free assay system. In yet another embodiment, antibodies generated to the translation products of the invention may be used in immunoprecipitation assays.
The present invention may also be used to screen for Pet-1 binding sites in genomic DNA. In, one embodiment cDNA encoding the invention may be used in microchip assays. The present invention contemplates a method of screening, comprising: a) providing in any order: i) a first solid support (e.g. microchip) comprising DNA from a DNA library of the species to be examined and ii) a peptide, or portion thereof, encoded by the DNA of SEQ ID NO:1; b) contacting said microassay microchips with said peptide under conditions such that hybridization can take place.
The present invention may also be used to identify new transcription factors that function in central serotonin 5-HT neurons. In one embodiment, antibodies generated to translation products of the invention may be used in immunoprecipitation experiments to isolate novel transcription factors that interact with Pet-1. In another embodiment, the invention may be used to generate fusion proteins that could also be used to isolate novel transcription factors or other interactive proteins. In yet another embodiment, screens may be conducted using the yeast two-hybrid system.
The present invention may also be used to identify new homologs of Pet-1 or natural mutations thereof. The present invention contemplates screening for homologs using standard molecular procedures. In one embodiment screens are conducted using Northern and Southern blotting.
The present invention contemplates a method of screening a compound, said method comprising: a) providing in any order: i) a first group of cells comprising a recombinant expression vector, wherein said vector comprises at least a
Deneris Evan Samuel
Fyodorov Dmitry Viktor
Hendricks Timothy John
Carlson Karen Cochrane
Medlen & Carroll, PLC LLP
Mitra Rita
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