Worm Breeder's Gazette 9(3): 116
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
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}
