This project has as its goals, to obtain and evaluate the structure of genomic DNA, particularly that associated with neurotransmitter receptor multigene families and neurological disorders, and to advance DNA cloning and sequencing strategies which will permit DNA sequencing on the megabase level. Successful strategies have been developed using two Applied Biosystems 370A Automated DNA Sequencers which currently allow up to 16 Kilo bases of DNA to be sequenced per day. Standard sequencing runs of over 500 bases/template are routine with greater than 99% accuracy. These techniques have been successfully applied to sequencing a number of neurotransmitter receptor genes (see project NS 02710-03). These procedures are being applied to further members of neurotransmitter receptor multigene family and to human chromosonal regions associated with genetic based neurological disorders. Areas of development include 1) the preparation of DNA templates for sequencing 2) the automation of template production 3) the automation of DNA sequence reactions 4) the extension of automated DNA sequence analysis to permit longer """"""""reads"""""""" per DNA template, and 5) the further development of computer-assisted analysis of DNA and protein sequence to determine DNA and protein structure. 1) Two major classes of neurotransmitter receptor gene families have been identified. The first includes the nicotinic acetylcholine, GABA/Benzodiazepine, and glycine receptors. The second major receptor family includes the adrenergic, muscarinic cholinergic, serotonin, substance k, receptors as well as the entire subfamily of opsins including bacteriorhodopsin.
The aim of this project is to obtain the primary structure of receptors in these families. 2) Genetic basis of neurological disorders: There are a number of neurological diseases that are caused to one degree or another by an inherited defect. Some of these diseases, such as neurofibromatosis are relatively common. The use of restriction fragment length polymorphisms has made it possible in theory to map any inherited disease to a small region of the genome. Considerable work has been done to map the regions involved in seal neurological diseases including neurofibromatosis I, neurofibomatosis II, Friedreichs ataxia, Huntington's disease and adrenoleukodystrophy. Our goal is to work with other NINCDS laboratories to map, clone and sequence the regions responsible for these diseases.