title: Identification of clones containing DNA complementary to phenylethanolamine N-methyltransferase mRNA.
abstract: Specific poly(A)mRNA for phenylethanolamine N-methyltransferase was isolated from bovine adrenal medulla by immunoprecipitation of polysomal mRNA with antibodies to bovine adrenal phenylethanolamine N-methyltransferase. Antibody-polysome complexes were recovered by Protein A Sepharose affinity chromatography. Phenylethanolamine N-methyltransferase mRNA, enriched 50-fold as judged by quantitative immunoprecipitation of translation products, was used as a template for the synthesis of complementary DNA (cDNA). Double-stranded cDNA was tailed with deoxycytosine and inserted into the Pst 1 site of poly(dG)-tailed plasmid pBR322. The resultant recombinant plasmids were used to transform competent E. coli strain 294. Tetracycline-resistant ampicillin-sensitive clones were screened by positive hybridization selection, and preliminary screening identified 2 out of 36 clones containing phenylethanolamine N-methyltransferase cDNA inserts. One phenylethanolamine N-methyltransferase cDNA insert was isolated from the plasmid DNA by digestion with Pst 1 and was found to be approximately 350 base-pairs in length. Northern blot analysis revealed that this phenylethanolamine N-methyltransferase cDNA probe strongly hybridized to an RNA species of approximately 1100 nucleotides.
authors.affiliation: Laboratory of Neurobiology, Cornell University Medical College, 1300 York Avenue, New York, NY 10021, U.S.A.
authors.forename: E E
authors.initials: EE
authors.lastname: Baetge
authors.forename: H M
authors.initials: HM
authors.lastname: Moon
authors.forename: B B
authors.initials: BB
authors.lastname: Kaplan
authors.forename: D H
authors.initials: DH
authors.lastname: Park
authors.forename: D J
authors.initials: DJ
authors.lastname: Reis
authors.forename: T H
authors.initials: TH
authors.lastname: Joh