Isolation and characterization of mutants for the vesicular acetylcholine transporter gene in Drosophila melanogaster

J Neurobiol. 2000 Feb 5;42(2):161-71.

Abstract

The Drosophila vesicular acetylcholine transporter gene (Vacht) is nested within the first intron of the choline acetyltransferase gene (Cha). To isolate Vacht mutants, we performed an F(2) genetic screen and identified mutations that failed to complement Df(3R)Cha(5), a deletion lacking Cha and the surrounding genes. Of these mutations, three mapped to a small genomic region where Cha resides. Complementation tests with a Cha mutant allele and rescue experiments using a transgenic Vacht minigene have revealed that two of these three mutations are nonconditional lethal alleles of Vacht (Vacht(1) and Vacht(2) ). The other is a new temperature-sensitive allele of Cha (Cha(ts3) ). Newly isolated Vacht mutants were used to reexamine the existing Cha mutations. We found that all deficiencies uncovering Cha also lack Vacht function, reflecting the nested organization of the two genes. The effective lethal phase for Vacht(1) is the embryonic stage, whereas that for Vacht(2) is the larval stage. Viable first-instar larvae homozygous for Vacht(2) showed reduced motility. Adult flies heterozygous for Vacht mutations were found to have defective responses in the dorsal longitudinal muscles following high-frequency brain stimulation. Since cholinergic synapses have been shown to be involved in the giant fiber pathway that mediates this response, the result suggested that reduction in the Vacht activity to 50% causes an abnormality in cholinergic transmission when stressed by a high-frequency stimulus.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alleles
  • Animals
  • Blotting, Western
  • Brain / enzymology
  • Carrier Proteins / genetics*
  • Carrier Proteins / physiology
  • Choline O-Acetyltransferase / metabolism
  • Drosophila Proteins
  • Drosophila melanogaster / genetics*
  • Drosophila melanogaster / physiology
  • Electrophysiology
  • Female
  • Genes, Lethal
  • Heterozygote
  • Larva / genetics
  • Larva / physiology
  • Male
  • Membrane Transport Proteins*
  • Motor Activity / genetics
  • Muscles / enzymology
  • Mutagenesis
  • Nerve Fibers / enzymology
  • Nerve Fibers / physiology
  • Synapses / genetics
  • Synapses / physiology
  • Temperature
  • Vesicular Acetylcholine Transport Proteins
  • Vesicular Transport Proteins*

Substances

  • Carrier Proteins
  • Drosophila Proteins
  • Membrane Transport Proteins
  • VAChT protein, Drosophila
  • Vesicular Acetylcholine Transport Proteins
  • Vesicular Transport Proteins
  • Choline O-Acetyltransferase