The behavior of the temperature sensitive allele
cha-1 (
cn101) is normal at 16 C but causes paralysis at 30 C. The choline acetyltransferase (ChAT) activity in crude extracts from the mutant is only 3 ,' of the wild-type at 16 C. We have characterized further the molecular properties of the enzyme. The mutant ChAT is very sensitive to temperature the activity is not detectable at 20 C, although the activity of the wild- type enzyme is maximal at 25 C. Under the higher concentration of salts, the wild-type ChAT is activated, but the mutant enzyme is inhibited. The mutant ChAT is much more sensitive to sulfhydryl blocking reagents tested than the wild-type enzyme. The mutant ChAT, therefore, is considered to lose the normal conformation depending u w n changes in the physical environment. Levels of acetylcholine (ACh), extracted with formic acid-acetone mixture, were determined with microradiometric assay (McCaman and Stetzler, J. Neurochem. 28 669 '77). As summarized below, the level of ACh in
cn101 is kept corresponding to the level in wild type when the animals were cultured at 16 C. We are especially interested in profiles of pool size of ACh in other
cha-1 and
unc-17 mutations, because a correlation is not observed as to the ChAT activity versus ACh in some alleles. {Figure 1} We are initiating some trials of seeking clones containing the
cha-1 gene regions. We were provided ca 2.2 kb cDNA clone for Drosophila ChAT by N.Itoh (the City of Hope. CA and now Kyoto Univ.). The coding region of the sequence spans 728 amino acids, which is 50-100 amino acids more than is required for an average Mr 67,000 protein. N2 DNA digested with EcoRl or Hind m did not hybridize with the Drosophila probe even under a mild condition (5 X SSC, 30 ) formamide, 42 C hybridization), although DNA from the Bergerac strain (CGC) showed weak signals at 3.2kb for EcoRl digestion, and 3.6kb and 5.7kb for Hind m digestion. As second trial to clone these regions via Tc1 tagging, we started to isolate spontaneous trichlorfon (TCF)-resistant mutants from the Beraerac and 4 mutator strains. RW7097 and RW7464 were kindly provided by D. Moerman and R.H. Waterston through I. Mori who informed us the detailed manual for Tc1 tagging with the strains. TR403 and TR679 were generously provided by Phil. Anderson. Animals were grown on 10 cm- agar plates containing 4-fold higher concentration of bactopeptone than normal NGM. The animals were transferred immediately before food exhaust to screening plates containing of 0.1 mM TCF. From 150 screening plates with the Bergerac strain, 8 mutants and from 150 screening plates with the RW7097, 16 mutants were found. Movement of most revertants is either normal or marginal in uncoordination but 4 mutants showed marked uncoordination. Screening with other mutator strains is in progress. In parallel with the work, as third trial for cloning, we are going on obtaining pure ChAT. Initial procedures for the purification are based on the modified method developed by Rand and Russell (J. Neurochem. 44 198 '85). By rechromatography of Affi-Gel Blue after hydroxylapatitetreatment, nearly pure ChAT was obtained (below). Although recovery of ChAT from C. ed, it may be possible to produce antisera with microgram quantity of the protein if we do in vitro immunization method. {Figure 2}