The human choline acetyltransferase gene encodes two proteins.

Both 2,500- and 6,000-nucleotide (nt) mRNAs are generated by alternative splicing of the primary transcript from the human gene for choline acetyltransferase (ChAT), the 68-kDa enzyme that synthesizes acetylcholine. In vitro translation of cRNA derived from a clone of the 2,500-nt mRNA produced a protein with ChAT activity demonstrating that this transcript encodes the human ChAT enzyme. An antibody directed against a unique amino acid sequence predicted from the 6,000-nt ChAT gene transcript identified a 27-kDa protein on immunoblots of human nucleus basalis proteins. This protein was further shown to cross-react with antibodies prepared against the 68-kDa human ChAT enzyme. Gel-filtration chromatography of human nucleus basalis proteins demonstrated that the 27-kDa protein does not have ChAT activity, which eluted as a single peak coincident with that of the 68-kDa enzyme. The 27-kDa protein was, however, shown to colocalize with the ChAT enzyme in cholinergic neurons of the human spinal cord using immunohistochemical techniques.

Two mRNAs are transcribed from the human gene for choline acetyltransferase.

The product of the choline acetyltransferase (ChAT) gene is the enzyme that synthesizes the neurotransmitter acetylcholine. A 14.4-kb portion of the human ChAT gene contains 7 exons, which are estimated to comprise approximately one-third of the human protein coding sequence by comparison with porcine ChAT mRNA. Two of the exons were used to identify polyadenylated human ChAT gene transcripts on Northern blots. An exon with 84% identity to the region of porcine ChAT mRNA that codes for the amino terminus of the corresponding protein detected 6,000- and 2,300-nucleotide mRNAs in RNA isolated from human CHP134 neuroblastoma cells. Only the 2,300-nucleotide mRNA was detected by a second probe containing an exon with 96% identity to porcine ChAT mRNA in the domain that encodes amino acids 204-263 of the predicted porcine ChAT protein. Further evidence that two species of human mRNA are produced from the human ChAT gene was obtained from nuclease protection assays using an antisense RNA probe prepared from a human ChAT cDNA clone. Total RNA isolated from either CHP134 cells or adult human nucleus basalis protected 525- and 400-nucleotide fragments of this probe, confirming the presence of two species of RNA that differ by the inclusion of an internal exon. cDNA clones of each of these transcripts have been isolated. Their sequences suggest that the 2,300-nucleotide mRNA encodes enzymatically active human ChAT, while translation of the 6,000-nucleotide mRNA would be terminated prematurely by a shift in the reading frame. These results indicate that a complex pattern of transcription produces two mRNAs with different coding potentials from the human ChAT gene.

Effect of a low-tryptophan diet as an adjuvant to conventional neuroleptic therapy in schizophrenia.

Eleven patients with DSM-III-R schizophrenia were entered into a 4-day tryptophan (TRP)-deficient diet. The diet lowered total plasma TRP levels in all patients; during the diet phase, there was a greater than 50% reduction in mean total plasma TRP levels from the pre-diet phase. The low-TRP diet improved performance on the Stroop Color and Word Test. These data are especially intriguing in view of the suggestion that a deficit in color-word naming is related to frontal lobe dysfunction and the possible occurrence of frontal lobe abnormalities in patients with schizophrenia. Interestingly, depressive symptomatology did not emerge on the TRP-deficient diet, despite the lowering of total plasma TRP levels. There were statistically significant improvements noted on objective ratings of the severity of psychotic symptomatology; however, these statistical improvements were without obvious clinical significance, as the magnitude of the changes on the behavioral ratings were minimal. The results of this study suggest that there might be some adjuvant potential for a low-TRP diet in the treatment of schizophrenia, and that schizophrenia or antipsychotic medications might offer some protection against the depressive effects of a TRP-deficient diet.

Expression of mouse cytochrome P450IA1 cDNA in repair-deficient and repair-proficient CHO cells.

Recombinant DNA techniques have been used to develop Chinese hamster ovary cell lines for studying chemically induced genotoxicity. These cell lines express a specific cytochrome P450 isozyme responsible for the metabolism of polycyclic aromatic hydrocarbons and exhibit defined differences in DNA repair capacity. A bacterial gene (neo) conferring resistance to gentamicin was inserted into the pcD expression vector containing the mouse cytochrome P1450 (P450IA1) cDNA to facilitate the selection of transformed cells. This plasmid was introduced into the nucleotide-excision-repair-deficient UV5 cell line by electroporation. Transformed clonal isolates expressing the P1450 cDNA were identified by differential cytotoxicity assays using benzo[a]pyrene (B[a]P). One such clone, termed UV5P1, was mutagenized with ethyl methanesulfonate and selected for resistance to killing by UV radiation to derive a repair-competent clone that expresses similar P1450 activity to that of the parental cell line. Two repair-competent clones were selected and called 5P1R1 and 5P1R3. The resulting cell lines (UV5P1, 5P1R1, and 5P1R3) expressed arylhydrocarbon hydroxylase activity. UV5P1 and 5P1R3 were compared in terms of cytotoxicity and mutagenicity after exposure to B[a]P. Induced mutations were measured at the adenine phosphoribosyltransferase (aprt) and hypoxanthine guanine phosphoribosyltransferase (hprt) loci. Repair-deficient UV5P1 cells were killed by B[a]P at concentrations below 0.1 microM, while the repair-proficient 5P1R3 cells showed no toxicity up to 60 microM. Mutation induction at both loci was also much more efficient in UV5P1 cells. These new cell lines provide a more sensitive system that can be used in a battery of assays to evaluate the genotoxicity of chemicals requiring P450IA1 metabolic activation and to assess the role of DNA repair in modulating the biological effects of DNA damage.