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...

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Published inNeurochemical research Vol. 24; no. 8; pp. 1081 - 1087
Main Authors WEIYA WANG, KITAMOTO, T, SALVATERRA, P. M
Format Journal Article
LanguageEnglish
Published New York, NY Springer 01.08.1999
Springer Nature B.V
Subjects
Alleles
Animals
Base Sequence
Biochemistry. Physiology. Immunology
Biological and medical sciences
Choline O-Acetyltransferase - genetics
Choline O-Acetyltransferase - metabolism
DNA Primers
Drosophila - enzymology
Drosophila melanogaster
Fundamental and applied biological sciences. Psychology
Heterozygote
Homozygote
Insecta
Invertebrates
Mutation
Physiology. Development
Receptors, Cholinergic - metabolism
RNA, Messenger - genetics
Synaptic Transmission
Temperature
Xenopus
Acyltransferases
Temperature
Enzyme
Insecta
Transferases
Drosophila
Choline O-acetyltransferase
Drosophilidae
Sensitivity
Arthropoda
Mutation
Invertebrata
Diptera
Cholinergic transmission
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Summary: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.
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ISSN:0364-3190
1573-6903
DOI:10.1023/A:1021021213625