Molecular characterization of a novel gene, dtr, which encodes a protein involved in neurotransmitter distribution and recycling
Abstract
We have cloned a novel gene, dtr (defective transmitter recycling), in Drosophila. The dtr mutant phenotype is characterized by the absence of the on-/off-transient components of the electroretinogram (ERG) and by an abnormal distribution of the neurotransmitter, histamine, in photoreceptors. The protein encoded by this gene, DTR, though not an integral membrane protein, seems to be involved in filling synaptic vesicles with neurotransmitter. The dtr locus was mapped by deficiency mapping and cloned using a positional cloning approach. The full-length dtr cDNA clone is 4739 bps. The rescue of the mutant phenotype by P element-mediated germline transformation using the full-length dtr cDNA unequivocally showed that the isolated cDNA clone corresponds to the dtr gene. The dtr transcript has a single open reading frame (ORF) encoding a protein of 1483 amino acids. The protein has no significant homology to any known protein except for a 200 amino acid region at the N-terminus which contains Leucine Rich Repeats (LRR). Various computer searches found several other motifs, none of which was helpful in predicting the possible function of the DTR protein. Since DTR does not have a transmembrane region or signal sequence, it is presumed to be a cytosolic protein. We postulate that the DTR protein is involved in the packaging of histamine into synaptic vesicles based on the following characteristics of the mutants. (1) Although the total content of neurotransmitter, as measured by HPLC, was normal, histamine release by a K+-induced depolarization was drastically reduced in the dtr mutants. (2) The dtr mutant phenotypes were age and light dependent, suggesting that the mutant phenotype increases in severity with synaptic activity. (3) dtr mutants were very slow to recover from the effects of previous stimuli, suggesting that they are defective in providing a pool of useable filled vesicles available for sustained activity. (4) Neurotransmitter is taken up by the synaptic endings normally but is inefficiently utilized for synaptic activity. (5) The location and approximate number of synaptic vesicles, determined by anti-CSP antibody staining, was normal in the synaptic region, but the transmitter was abnormally distributed throughout the photoreceptor cell body in the mutants.
Degree
Ph.D.
Advisors
Pak, Purdue University.
Subject Area
Neurology|Molecular biology
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