Drosophila choline acetyltransferase temperature-sensitive mutants.
We used the reverse transcription-polymerase chain reaction (RT-PCR) to amplify choline acetyltransferase (ChAT) mRNA fragments from two temperature-sensitive alleles of Drosophila melanogaster, Cha(ts1) and Cha(ts2). Single base substitutions in the mutants (T1614A in Cha(ts1) and G1596A in Cha(ts2)) would result in amino acid changes for ChAT protein (Met403Lys in Ch(ts1) and Arg397His in Cha(ts2)). These base substitutions were confirmed in mRNA extracted from homozygous mutants using a Single Nucleotide Primer Extension assay (SNuPE) and are sufficient to produce thermolabile enzyme. Our results indicate that these temperature-sensitive mutants are point mutations in the structural gene for ChAT. Using a quantitative SNuPE assay we also show that similar levels of Cha(ts) and wild type transcripts are present in heterozygous flies (Cha(ts1)/+ and Cha(Ts2)/+) at both restrictive and permissive temperatures. This contrasts with RNase protection assays of ChAT mRNA in homozygous mutant animals where the levels of mutant mRNA decrease at restrictive temperature.
Wang
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Division of Neurosciences, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA.
Kitamoto
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Salvaterra
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PM
D000483:Alleles; D000818:Animals; D001483:Base Sequence; D002795:Choline O-Acetyltransferase / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D004330:Drosophila / Q000201:enzymology*; D006579:Heterozygote; D006720:Homozygote; D009154:Mutation; D012333:RNA, Messenger / Q000235:genetics; D011950:Receptors, Cholinergic / Q000378:metabolism; D009435:Synaptic Transmission; D013696:Temperature; D014981:Xenopus
D017931:DNA Primers; D012333:RNA, Messenger; D011950:Receptors, Cholinergic; D002795:Choline O-Acetyltransferase