Partial cloning of the rat choline acetyltransferase gene and in situ localization of its transcripts in the cell body of cholinergic neurons in the brain stem and spinal cord.
We have isolated recombinant lambda (lambda) phages which contain a part of the rat choline acetyltransferase (ChAT) gene. Restriction and Southern blot analyses using synthetic oligonucleotides indicate that these clones overlap one another and contain at least four exons which reside in 16.4 kb of sequence encoding from the middle to the 3' end, but not the 5'-region, of the rat ChAT gene. Partial sequence analyses revealed that the clones contain an exon whose nucleotide sequence corresponds to a highly conserved region of ChAT during evolution. RNase protection mapping experiments show that sequences represented by this exon are expressed at high levels in the spinal cord of adult rats and at low but detectable levels in PC12 cells. By using the genomic sequences, including the exon, as a hybridization probe, we have detected ChAT mRNAs in situ in rat tissues. In situ hybridization experiments using radioactive and non-radioactive probes revealed that cholinergic motoneurons in the spinal cord, the laterodorsal tegmental nucleus as well as the hypoglossal nucleus in the brain stem were labeled, suggesting that the genomic sequence can be used as a probe to measure the ChAT mRNA levels in those cholinergic neurons. The results also indicate that the non-radioactive method gives a better resolution in localizing the expression of ChAT transcripts in the cytoplasm of cholinergic neurons.
Division of Neurogerontology, Ethel Percy Andrus Gerontology Center, University of Southern California, Los Angeles 90089-0191.
N
N
Mori
Y
Y
Tajima
H
H
Sakaguchi
D J
DJ
Vandenbergh
H
H
Nawa
P M
PM
Salvaterra
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001933:Brain Stem / Q000166:cytology / Q000201:enzymology*; D002795:Choline O-Acetyltransferase / Q000235:genetics*; D003001:Cloning, Molecular; D004790:Enzyme Induction; D005091:Exons; D005796:Genes; D017403:In Situ Hybridization; D008297:Male; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics*; D009474:Neurons / Q000201:enzymology*; D051381:Rats / Q000235:genetics*; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000032:analysis; D013116:Spinal Cord / Q000166:cytology / Q000201:enzymology*; D013552:Swine / Q000235:genetics; D014158:Transcription, Genetic
D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D002795:Choline O-Acetyltransferase