Title

Multiple proteins are encoded by a single human choline acetyltransferase gene

Date of Award

1993

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

First Committee Member

William L. Strauss, Committee Chair

Abstract

Acetylcholine is synthesized by the enzyme choline acetyltransferase (acetyl CoA: choline-O-acetyltransferase (EC 2.3.1.6), ChAT). Four aspects of ChAT gene expression were studied: (1) the characterization of a portion of the human ChAT gene; (2) the regulation of ChAT mRNA by nerve growth factor (NGF) in rat basal forebrain cultures; (3) the characterization of two human ChAT mRNAs; (4) the identification of the translation products of two human ChAT mRNAs. A 14.4 kilobase genomic clone of human ChAT was found to contain seven exons of the human ChAT gene. An exon-specific probe of the human ChAT gene was used to demonstrate that the relative abundance of ChAT mRNA was increased in rat basal forebrain cultures treated with NGF. In humans, the results of Northern blotting, nuclease protection, and cDNA cloning experiments demonstrated that 6000 nt and 2300 nt mRNAs are transcribed from a single ChAT gene. A comparison of the sequences of the cDNAs derived from the 2300 nt and 6000 nt human ChAT mRNAs revealed that the difference between the two mRNAs within the cloned region was the presence (2300 nt) or absence (6000 nt) of an internal protein coding exon. The omission of this exon in the 6000 nt mRNA causes a shift in the frame of translation predicting the translation of a truncated protein with a unique nine amino acid carboxylic terminus. These results indicated that each human ChAT mRNA encoded a different protein. Polyclonal antibodies were raised against peptides specific for each of the predicted translation products and used to detect the corresponding proteins by immunoprecipitation and Western blot analysis. The antiserum directed against the translation product of the 2300 nt mRNA identified a 68 kilodalton protein. Antibodies directed against the nine amino acid carboxylic terminus predicted from the 6000 nt mRNA detected a 27 kilodalton protein in human cholinergic tissue. These findings indicate that two proteins are encoded by a single human ChAT gene.

Keywords

Biology, Molecular; Biology, Neuroscience

Link to Full Text

http://access.library.miami.edu/login?url=http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:9331492