Gene expression of mouse choline acetyltransferase. Alternative splicing and identification of a highly active promoter region.

H Misawa,K Ishii,T Deguchi

doi:10.1016/s0021-9258(19)88714-8

Abstract

Seven types of mRNA for choline acetyltransferase that differ in the 5'-noncoding region were identified in the mouse spinal cord by cDNA cloning and polymerase chain reaction. Among these transcripts, the M-type mRNA corresponding to the previously cloned mouse cDNA was most abundant in the spinal cord of mouse. A mouse genomic DNA clone containing the 5'-region of choline acetyltransferase mRNA was isolated and sequenced. Comparison of the sequences between the cDNAs and the genomic DNA revealed that the different mRNA species were transcribed from different promoter regions and produced by differential splicing. Two murine cholinergic cell lines, NS20Y and NG108-15, were shown to express the M-type mRNA almost exclusively, and were therefore used to study transcription of M-type mRNA. Fragments of the 5'-region of choline acetyltransferase gene were ligated with chloramphenicol acetyltransferase reporter gene and introduced into cultured cells. The fragment from -2752 to +46, which contained the M-type exon, a TATA-box like element upstream of the M-type exon, and the downstream intron, induced a significant expression of CAT activity in neuronal but not in non-neuronal cell lines. This result indicates that this region of choline acetyltransferase gene contains elements that regulate neuron-specific expression of choline acetyltransferase activity. However, there was no parallel correlation between reporter gene expression in the transfected cells and intrinsic choline acetyltransferase activity in these neuronal cell lines. Possible mechanisms that would explain this observation are discussed.

Highlights

Seven types of mRNA for choline acetyltransferase that differ in the 5'-noncoding region were identified in the mouse spinal cord by cDNA cloning and polymerase chain reaction
Cloning of 5"Regions of Choline AcetyltransferasecDNAsA cDNA library was constructed from the poly(A)+ RNA of mouse spinal cord usinag 17-mer oligonucleotide primer (Fig. 1, Rev-1 ) that was complementary to position6s42-658 of the coding region of mouse choline acetyltransferase cDNA [7]
Analysis of choline acetyltransferase mRNAby cDNAcloning and PCR amplification identified seven types of mRNA in the mouse spinal cord that differed in the 5"noncoding region

Summary

ALTERNATIVE SPLICING AND IDENTIFICATIONOFAHIGHLY ACTIVE PROMOTER REGION*

Seven types of mRNA for choline acetyltransferase that differ in the 5'-noncoding region were identified in the mouse spinal cord by cDNA cloning and polymerase chain reaction. Choline acetyltransferase activity in cultured motoneurons from the mouse spinal cord was markedly enhanced by co-culture with skeletal myotubes [4] It is not known how transcription of choline acetyltransferase gene is regulated under these conditions. We have found that there are five types of choline acetyltransferase mRNA that differ in the 5"noncoding region, and that"type mRNA corresponding to the previously cloned mouse cDNA is the most abundant type in the spinal cord of rat.'. Assay of expressed CAT activity indicated that the5"region of the choline acetyltransferase gene contains elements that regulate high expression of the reporter gene in neuronal, but not in non-neuronalcell lines. PCR products (5 p l ) or 5'-RACE products (2 pl) were separated on a 2.5 or 3% NuSieve GTG agarose (FMC Bioproducts) gel containing ethidium bromide (EtBr, 0.5 pg/ml) and transferred to a

RESULTS

Mouse Choline Acetyltransferase Gene

IS I

DISCUSSION

Connective tissue