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Drosophila Choline Acetyltransferase Temperature-Sensitive Mutants

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Abstract

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 Cha 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.

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Authors and Affiliations

  1. Division of Neurosciences, Beckman Research Institute of the City of Hope, 1450 E. Duarte Rd., Duarte, CA, 91010

    Weiya Wang, Toshihiro Kitamoto & Paul M. Salvaterra

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  1. Weiya Wang
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  2. Toshihiro Kitamoto
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  3. Paul M. Salvaterra
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Wang, W., Kitamoto, T. & Salvaterra, P.M. Drosophila Choline Acetyltransferase Temperature-Sensitive Mutants. Neurochem Res 24, 1081–1087 (1999). https://doi.org/10.1023/A:1021021213625

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