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...

Microsatellites are common repeated sequences, which are useful as genetic markers and lack any clearly established function. In a previous study we suggested that an intronic polymorphic TCAT repeat in the tyrosine hydroxylase (TH) gene, the microsatellite HUMTH01, may regulate transcription. The TH gene encodes the rate-limiting enzyme in the synthesis of catecholamines, and the microsatellite HUMTH01 has been used in genetic studies of neuropsychiatric and cardiovascular diseases, in which disturbances of catecholaminergic neurotransmission have been implicated. HUMTH01 alleles associated with these diseases act as transcriptional enhancers when linked to a minimal promoter and are recognized by specific nuclear factors. Here we show that allelic variations of HUMTH01 commonly found in humans have a quantitative silencing effect on TH gene expression. Two specific proteins, ZNF191, a zinc finger protein, and HBP1, an HMG box transcription factor, which bind the TCAT motif, were then cloned. Finally, allelic variations of HUMTH01 correlate with quantitative and qualitative changes in the binding by ZNF191. Thus, this repeated sequence may contribute to the control of expression of quantitative genetic traits. As the HUMTH01 core motif is ubiquitous in the genome, this phenomenon may be relevant to the quantitative expression of many genes in addition to TH.

We have previously shown that the phorbol ester, TPA, which activates protein kinase C, causes, in PC12 cells, a transcriptional activation of tyrosine hydroxylase (TH), the key enzyme in catecholamine synthesis. The study has now been extended to examine the processes that underlie this transcriptional stimulation and, in addition, to seek whether similar mechanisms are involved in long-term trans-synaptic induction of the TH gene in adrenal medullae of rats that have been given a single injection of reserpine. In both systems, it was found that the induction of c-fos gene transcription was associated with that of the TH gene but with different kinetics. The promoter of the TH gene contains (at position -207/-200) a sequence (TGATTCA) which differs from the consensus TRE or AP-1 site (TGACTCA) by one nucleotide. Experiments were carried out to investigate whether the AP-1 protein complex which is known to contain Fos and Jun binds to the putative TRE region of the TH promoter. In the gel shift assays, the nuclear protein extracts derived from TPA-treated PC12 cells and from AM of reserpine injected rats displayed a higher magnitude of binding to a 25-mer TRE-TH oligonucleotide as compared to controls. The results showed that the behaviour of TRE-TH was atypical in that two retarded complexes (A and B) were observed, which were displaced by specific competitors. Trans-activation experiments with plasmids TRE-TH/TK/CAT and -754/-19 TH/pUC18-CAT in PC12 cells showed an increase in CAT activity in response to TPA that correlates with the previously observed increase in TH transcriptional activity by TPA.(ABSTRACT TRUNCATED AT 250 WORDS)

The role played by protein kinase C (PKC) in TH gene regulation was investigated at transcriptional and post‐transcriptional levels using PC12 cells. The cells were treated with the phorbol ester TPA, which not only activates PKC but also causes down‐regulation. PKC levels were monitored by [3H]PDBU binding assay and by using an anti‐PKC antibody that detected intact PKC (79 kd) as well as its catalytic and regulatory domains. The [3H]PDBU binding to the membrane‐associated PKC increased within 15‐30 min of TPA treatment; thereafter total cellular [3H]PDBU binding decreased to a minimum of 20% of the control at 8 h. The rate of decrease in binding was greater than the decrease in the intensity of the staining of PKC holo enzyme visualized by anti‐PKC antibody. TH mRNA levels, measured over the same time period, rose within 15 min of TPA treatment to peak at 4 h and subsequently declined below control level, paralleling the depletion of PKC. If cells depleted of PKC were reincubated in the normal medium, a recovery in PKC level was seen and, in parallel, TH mRNA levels increased to above control level. Furthermore, if down‐regulation of PKC was prevented by incubating the cells with the protease inhibitor leupeptin, a decrease beyond control level in TH mRNA was not observed. TPA rapidly induced TH gene transcription; a maximal increase of two‐fold was observed at 15 min, but the transcriptional rate then declined although it did not decrease beyond control values after 8 and 24 h of TPA treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

A full length dopamine‐beta‐hydroxylase (DBH) cDNA clone was isolated from a human pheochromocytoma lambda gt11 library. Both structural and functional evidence confirms the authenticity of the clone: (i) antibodies selected with fusion proteins generated by positive clones precipitate DBH activity, (ii) the sequence of three internal DBH tryptic peptides are included in the deduced DBH sequence, (iii) the previously reported N‐terminal 15 amino acids of bovine DBH exhibits a nearly complete identity with that predicted for human DBH. The polypeptide chain of DBH comprises 578 amino acids corresponding to an unmodified protein of 64 862 daltons and is preceded by a cleaved signal peptide of 25 residues. DBH exists in both membrane‐bound and soluble forms. The hydropathy plot reveals no obvious hydrophobic segment, except the signal peptide. S1 mapping analysis indicates no diversity in the 5′ and 3′ extremities of the DBH mRNA. Taken together with available biochemical data, these observations suggest that the membrane attachment of DBH probably results from a post‐translational modification, glypiation being the most likely candidate. Comparative amino acid sequence analysis establishes that DBH shares no homology with the other catecholamine synthesizing enzymes, tyrosine hydroxylase and phenylethanolamine‐N‐methyl transferase.

A single injection of reserpine causes a long lasting enhancement of the activity of tyrosine hydroxylase (TH), the enzyme catalyzing the rate‐limiting step in the biosynthesis of catecholamines. A sensitive method has been developed to assay both TH mRNA level and enzyme activity in tissue from a single rat. The time course of the induction was analysed in adrenals, locus coeruleus and substantia nigra. In both locus coeruleus and adrenals reserpine caused respectively 4.2‐ and 4.5‐fold increase of TH mRNA which was maximal 2 days after drug injection. This increase is about twice that of the enzyme activity. No change was observed in substantia nigra. The effect lasted longer in locus coeruleus than in adrenal. In the latter, TH mRNA had almost returned to initial values at day 4 whereas at this time it is 3‐fold higher in locus coeruleus and still significant at day 18. This result suggests that induction of TH results from an enhanced transcription of the TH gene. The time course difference between locus coeruleus and adrenals is most likely to result from a difference in the stability of TH mRNA in the two structures.

GPR88, coding for a G protein-coupled orphan receptor that is highly represented in the striatum, is a strong functional candidate gene for neuropsychiatric disorders and is located at 1p22-p21, a chromosomal region that we have previously linked to bipolar disorder (BD) in the Sardinian population. In order to ascertain the relevance of GPR88 as a risk factor for psychiatric diseases, we performed a genetic association analysis between GPR88 and BD in a sample of triads (patient and both parents) recruited in the Sardinian and the Palestinian population as well as between GPR88 and schizophrenia (SZ) in triads from the Xhosa population in South Africa. We found a positive association between GPR88 and BD in the Sardinian and Palestinian triads. Moreover, we found a positive association between GPR88 and SZ in triads from the Xhosa population in South Africa. When these results were corrected for multiple testing, the association between GPR88 and BD was maintained in the Palestinian population. Thus, these results suggest that GPR88 deserves consideration as a candidate gene for psychiatric diseases and requires to be further investigated in other populations.

Many studies provide evidence that retinoic acid (RA), an endogenous derivative of vitamin A, plays a role in the development of the nervous system. We now report that RA controls the neurotransmitter phenotype of post-mitotic rat sympathetic neurons in cell culture. RA added to the culture medium increased the specific activity of choline acetyltransferase (ChAT) and the level of acetylcholine (ACh). Concomitantly, RA reduced the specific activities of two catecholamine synthetic enzymes, tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) and the level of norepinephrine (NE). After a 2 week treatment with 5 microM RA, ChAT was increased by 5-10 fold, whereas TH and DBH were decreased by 10-15 fold and 2-3 fold, respectively, as compared to sympathetic neurons grown in the absence of RA. The modulation of the activity of the three enzymes was dose-dependent and followed a similar time course. The decrease of TH expression was demonstrated to be due to a decreased number of TH molecules.