Complete coding sequence of rat tyrosine hydroxylase mRNA.

Abstract

Several clones specific for tyrosine hydroxylase [tyrosine 3-monooxygenase, L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2] have been identified from a rat PC12 library by using the previously characterized clone pTH-1. The most complete of these, pTH-51, is 1758 base pairs long and covers most of the length of the mRNA, including the entire coding and 3' untranslated region. The polypeptide has an estimated molecular weight of 55,903 and some of its characteristic features are discussed.Tyrosine hydroxylase [TyrOHase; tyrosine 3-monooxygenase; L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1. 14.16.2], the rate-limiting enzyme in the synthesis of catecholamines, has been intensively investigated because of its key role in the physiology of adrenergic neurones. The regulation of its expression is under developmental control (1) and its synthesis can be induced in vivo by nerve stimulation (2, 3) or by treatment with reserpine (4-6) or steroids (7). Also, multiple kinase activities may be involved in the short-term regulation of catecholamine biosynthesis by afferent activity (8-13).As an initial step to determine the molecular mechanism by which the gene is expressed, we have recently reported the identification of cloned recombinant cDNA encoding this enzyme (14).In this paper, we describe the selection of other cDNA clones specific for rat TyrOHase that cross-hybridize with the previously isolated pTH-1 clone (14). One clone contains the entire coding and 3' untranslated region of TyrOHase mRNA. From the nucleotide sequence of the cDNAs, we have deduced the entire amino acid sequence of this previously uncharacterized protein.MATERIALS AND METHODS Materials. Enzymes, except stated otherwise, were from Bethesda Research Laboratories. Reverse transcriptase was obtained from J. Beard (Life Sciences, St. Petersburg, FL). DNA polymerase I, terminal deoxynucleotidyltransferase from calf thymus, oligo(dT)-cellulose (T7), and the 17-base synthetic M13 primer were from P-L Biochemicals. Nitrocellulose filters were purchased from Millipore (HAWP type). [a-32P]dNTPs (>400 Ci/mmol; 1 Ci = 37 GBq) were purchased from the Radiochemical Centre.Construction and Isolation of TyrOHase cDNA Clones. Polyadenylylated mRNAs were obtained from PC12 cells as reported (15). Double-stranded cDNA was synthesized as in ref. 16 and fractionated in a 5-20% sucrose gradient. The longest molecules [>'600 base pairs (bp)] were inserted into the Pst I site of pBR322 by oligo(dGdC) tailing (17). The recombinant plasmids were used to transform Escherichia coli strain MC1061 (18) by the efficient procedure described by Hanahan (19). Colonies were plated at high density on 22-cm-square nitrocellulose filters overlying LB agar containing 15 ,ug of tetracycline per ml according to Grosveld (20). Approximately 50,000 recombinant clones were obtained from 5 ,g of poly(A)+ mRNA. Colonies on duplicate nitrocellulose filters were lysed as described by Thayer (21) and screened u...

Differential Role of Galanin Receptors in the Regulation of Depression-Like Behavior and Monoamine/Stress-Related Genes at the Cell Body Level

“…Oligonucleotides complementary to rat galanin nucleotides 152-199 (Vrontakis et al, 1987), tyrosine hydroxylase (TH) nucleotides 1441-1488 (Grima et al, 1985), tryptophan hydroxylase 2 (TPH2) nucleotides 489-536 (Walther et al, 2003), rat 5-HT 1A receptor nucleotides 762-809 and 1048-1095 (Albert et al, 1990) and 5-HT transporter (5-HTT) nucleotides 305-352 (Hoffman et al, 1991) were used to study mRNA expression levels in the LC (TH and galanin) and in the DR/MR (TPH2, galanin, 5HT 1A , and 5-HTT). Oligonucleotide probes were labeled with 33 P (dATP) (NEN) at the 3 0 -end using terminal deoxynucleotidyltransferase (Amersham, Amersham, UK) and purified using ProbeQuantt G-50 microcolumns (Amersham Pharmacia Biotech, Piscataway, NJ, USA).…”

A Centrally Acting, Anxiolytic Angiotensin II AT1 Receptor Antagonist Prevents the Isolation Stress-Induced Decrease in Cortical CRF1 Receptor and Benzodiazepine Binding

“…We synthesized one antisense oligonucleotide of 48-mer for the rat TH cDNA sequence (Lofstrand Labs Limited, MD), localized in nt 1562-1609 (Grima et al, 1985), and labeled the oligonucleotide with terminal deoxynucleotidyl transferase (Amersham) to a specific activity of 3-4 Â 10 8 dpm/mg. Each reaction was performed with 70 pmol of oligonucleotides in the presence of 70 mCi of [a-35 S]ATP (Amersham).…”

Neurotensin-deficient mice show altered responses to antipsychotic drugs

“…An NsiI͞EcoRI fragment spanning exon 4 (nucleotides 9522-10317) of the mouse NT͞N gene was subcloned into pGEM4 (Promega) that had been digested with PstI and EcoRI for use as a probe. Probes for human substance P (27), rat enkephalin (28), and rat tyrosine hydroxylase (29) were generated as described.…”