1985 |
Joh, Tong H |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Gene Regulation in Cholinergic and Adrenergic Neurons @ Weill Medical College of Cornell Univ
The present proposal is designed to elucidate molecular mechanisms governing the gene expression and regulation of enzymes involved in catecholamine and acetylcholine biosynthesis. These enzymes are tyrosine hydroxylase, dopadecarboxylase, dopamine B-hydroxylase, and phenylethanolamine N-methyltransferase for catecholamine synthesis, and choline acetyltransferase for the biosynthesis of acetylcholine. Changes in neurotransmitter enzyme activity in the central and peripheral nervous systems and adrenal gland, caused by drugs, hormones, nervous injury and other factors will be studied by determining RNA concentrations and transcriptional rates as a means to further understand the mechanisms which regulate gene expression. Further, the regulation of phenotypic and gene expression during the embryonic development will be investigated. To carry out these investigations, the following strategy is employed: (a) clonal cell lines of neural and chromaffin cell origins will be used as simple systems to investigate the mechanisms governing enzyme induction, since they are easy to manipulate and will allow us to obtain large amounts of mRNA and nuclei; (b) using recombinant DNA technology, complementary DNA to enzyme RNA will be synthesized; (c) sensitive molecular hybridization procedures, such as Dot blot hybridization and in situ hybridization histochemistry using radiolabeled enzyme-cDNA probes, will be used to determine the quantity of specific enzyme mRNA; (d) in order to determine the rate of enzyme-mRNA synthesis, mRNA transcription will be measured in isolated nuclei of neuronal or chromaffin cells; and, finally, (e) these studies will be extended to the central and peripheral nervous systems and adrenal medulla. Thus, the present studies will provide, for the first time, a detailed insight into how alterations in the pharmacological, hormonal and physiological environment of neurons and chromaffin cells affected changes in gene expression of neurotransmitter synthesizing enzymes.
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1 |
1985 |
Joh, Tong H |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Similar Gene Coding Sequences of Neurotransmitters @ Weill Medical College of Cornell Univ
We propose that the catecholamine synthesizing enzymes, tyrosine hydroxylase (TH), dopamine-B-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT), share common gene coding sequence(s). Conceivably, these enzymes are coded for genes that have evolved through duplication of a common ancestral precusor. Further, we propose to include in our hypothesis that tryptophan hydroxylase (TPH), which catalyses the first step in serotonin biosynthesis, is also coded for by a similar gene. Evidence for the evolution of the catecholamine and serotonin synthesizing enzyme genes through mutation or duplication of ancestral gene(s) can be gained by determining their molecular anatomy. In order to prove our hypothesis, we plan the following experiments: (1) Purification of TH, DBH, PNMT and TPH to homogeneity; (2) Production of specific antibodies to each enzyme; (3) Identification of amino acid sequence homology of each enzyme, by determining amino acid compostions and sequences of peptides obtained from each enzyme by chemical and proteolytic digestion; (4) Cloning of cDNA to TH, DBH, PNMT and TPH poly(A)mRNA; and (5) Examination of the organization and structure of the genes involved in catecholamine and serotonin biosynthesis. Determination of the structural relationships between the catecholamine and serotonin synthesizing enzyme genes will contribute to elucidation of the molecular mechanisms involved in the formation of common gene coding sequence(s), and will provide valuable insight into both developmental and evolutionary processes by which neurons express the dopaminergic, noradrenergic, adrenergic or serotonergic phenotype.
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1 |
1986 — 1998 |
Joh, Tong H |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. R37Activity Code Description: To provide long-term grant support to investigators whose research competence and productivity are distinctly superior and who are highly likely to continue to perform in an outstanding manner. Investigators may not apply for a MERIT award. Program staff and/or members of the cognizant National Advisory Council/Board will identify candidates for the MERIT award during the course of review of competing research grant applications prepared and submitted in accordance with regular PHS requirements. |
Structural Analysis of Catecholamine Enzyme Genes @ Burke Rehabilitation Ctr (White Plns,Ny)
Catecholamine neurotransmitter systems have been implicated in the patho-physiology of some central nervous system diseases including schizophrenia, manic depression, Parkinson's and Alzheimer's syndromes. The understanding of the expression and regulation of this important metabolic pathway is not only of biological interest but critical to exploring the fundamental basis of these disorders and more rationally approaching their diagnosis and therapy. Our strategy requires the structural analysis of the genes for all four catecholamine synthesizing enzymes in order to investigate the coordinate and differential regulation in vivo. We have isolated and characterized cDNAs for tyrosine hydroxylase, aromatic L-amino acid decarboxylase, dopamine B-hydroxylase and phenylethanolamine N-methyltransferase. These are now being used to study changes in mRNA expression under various physiological conditions. In addition, genomic clones for each of the enzymes are being identified and characterized with these molecular probes. The following experiments are proposed to accomplish these goals: 1. Definition of the basic structural elements of the catecholamine enzyme genes by isolation of genomic clones, mapping and sequence analysis; 2. Determination of those physiological regulators which influence the transcription of these genes in vivo using primary bovine chromaffin cells as a model; 3. Identification and characterization of the genetic elements responsible for cell-type specific and regulated expression of each enzyme gene by transfection assay and mutagenesis of putative regulatory elements; 4. Examination of the interaction of nuclear trans-acting factors with regulatory regions in the gene using footprinting and in vitro transcription assays; 5. Investigation of possible common elements and/or factors shared among the genes which underly coordinate regulation of the pathway using competition assays. These experiments will not only further our understanding of this important biosynthetic pathway but will provide the techniques and molecular tools to approach the purification of trans-acting factors regulating the tissue specific and regulated expression of the catecholamine enzyme genes. Their identification and characterization will be key to understanding this regulation at the molecular level.
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1 |
1987 — 1989 |
Joh, Tong H |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Molecular Neurobiology of Catecholamine Enzymes @ Burke Rehabilitation Ctr (White Plns,Ny)
catecholamines; neurophysiology; molecular biology; enzymes;
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0.909 |