Sequence of choline acetyltransferase temperature-sensitive mutants determined by the polymerase chain reaction.
Two temperature-sensitive alleles of Drosophila melanogaster, Cha(ts1) and Cha(ts2), have been previously identified and are thought to be point mutations in the structural gene for the neurotransmitter biosynthetic enzyme choline acetyltransferase. In order to clarify the molecular nature of these alleles and characterize the presumed amino acid substitutions, we have used the polymerase chain reaction to amplify choline acetyltransferase messenger ribonucleic acid fragments from both mutant genotypes. Amplified mutant complementary deoxyribonucleic acid was cloned and used to construct chimeric complementary deoxyribonucleic acid clones containing approximately two-thirds of the wild-type sequence which would code for N-terminal amino acids and one-third of the mutant sequence coding for the C-terminal amino acids. After in vitro translation of complementary ribonucleic acid produced from the chimeric complementary deoxyribonucleic acid clones, choline acetyltransferase activity was determined and shown to be thermolabile. Sequence analyses of these clones showed that one amino acid substitution due to single base substitution is crucial in each chimeric choline acetyltransferase complementary deoxyribonucleic acid to generate a thermolabile choline acetyltransferase product. The point mutations of the structural gene for choline acetyltransferase are thus confirmed and shown to regulate the thermolability of the enzyme produced by Cha(ts1) and Cha(ts2).
Tajima
Y
Y
Division of Neurosciences, Beckman Research Institute of the City of Hope, Duarte, CA 91010.
Salvaterra
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PM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002795:Choline O-Acetyltransferase / Q000235:genetics* / Q000378:metabolism; D003062:Codon; D004331:Drosophila melanogaster / Q000201:enzymology / Q000235:genetics; D005796:Genes; D005804:Genes, Lethal; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping; D013816:Thermodynamics
D003062:Codon; D015345:Oligonucleotide Probes; D002795:Choline O-Acetyltransferase