Bovine Tyrosine Hydroxylase Research Articles

Genetic diversity of tyrosine hydroxylase (TH) and dopamine b-hydroxylase (DBH) genes in cattle breeds

DNA from four cattle breeds was used to re-sequence all of the exons and 56% of the introns of the bovine tyrosine hydroxylase (TH) gene and 97% and 13% of the bovine dopamine β-hydroxylase (DBH) coding and non-coding sequences, respectively. Two novel single nucleotide polymorphisms (SNPs) and a microsatellite motif were found in the TH sequences. The DBH sequences contained 62 nucleotide changes, including eight non-synonymous SNPs (nsSNPs) that are of particular interest because they may alter protein function and therefore affect the phenotype. These DBH nsSNPs resulted in amino acid substitutions that were predicted to destabilize the protein structure. Six SNPs (one from TH and five from DBH non-synonymous SNPs) were genotyped in 140 animals; all of them were polymorphic and had a minor allele frequency of > 9%. There were significant differences in the intra- and inter-population haplotype distributions. The haplotype differences between Brahman cattle and the three B. t. taurus breeds (Charolais, Holstein and Lidia) were interesting from a behavioural point of view because of the differences in temperament between these breeds.

The bovine tyrosine hydroxylase gene associates in vitro with the nuclear matrix by its first intron sequence.

Recently we have shown that in vitro binding of the proximal part of the human tyrosine hydroxylase gene to the nuclear matrix is correlated with its transcriptional activity. The strongest binding potential was predicted by computing for the first intron sequence (Lenartowski & Goc, 2002, Neurosci Lett.; 330: 151-154). In this study a 16 kb fragment of the bovine genomic DNA containing the tyrosine hydroxylase gene was investigated for its affinity to the nuclear matrix. Only a 950 bp fragment encoding the distal part of the first intron, second exon and a few nucleotides of the second intron bound to the nuclear matrix. The binding was independent of the tissue-specific tyrosine hydroxylase gene activation. The fragment was subcloned and sequenced. Computer search pointed to one potential intronic matrix attachment region with two AP1-like sites embedded in the sequence. We conclude that even if the position of the matrix binding region is conserved among the tyrosine hydroxylase genes in mammals, its tissue specificity and/or function is not preserved or is achieved by different mechanisms.

The bovine tyrosine hydroxylase gene associates in vitro with the nuclear matrix by its first intron sequence.

Recently we have shown that in vitro binding of the proximal part of the human tyrosine hydroxylase gene to the nuclear matrix is correlated with its transcriptional activity. The strongest binding potential was predicted by computing for the first intron sequence (Lenartowski & Goc, 2002, Neurosci Lett.; 330: 151-154). In this study a 16 kb fragment of the bovine genomic DNA containing the tyrosine hydroxylase gene was investigated for its affinity to the nuclear matrix. Only a 950 bp fragment encoding the distal part of the first intron, second exon and a few nucleotides of the second intron bound to the nuclear matrix. The binding was independent of the tissue-specific tyrosine hydroxylase gene activation. The fragment was subcloned and sequenced. Computer search pointed to one potential intronic matrix attachment region with two AP1-like sites embedded in the sequence. We conclude that even if the position of the matrix binding region is conserved among the tyrosine hydroxylase genes in mammals, its tissue specificity and/or function is not preserved or is achieved by different mechanisms.

Ubiquitination of soluble and membrane-bound tyrosine hydroxylase and degradation of the soluble form.

Tyrosine hydroxylase (TH) demonstrates by two-dimensional electrophoresis a microheterogeneity both as a soluble recombinant human TH (hTH1) and as a membrane-bound bovine TH (bTHmem). Part of the heterogeneity is likely due to deamidation of labile asparagine residues. Wild-type (wt)-hTH1 was found to be a substrate for the ubiquitin (Ub) conjugating enzyme system in a reconstituted in vitro system. When wt-hTH1 was expressed in a coupled transcription-translation TnT(R)-T7 reticulolysate system 35S-labelled polypeptides of the expected molecular mass of native enzyme as well as both higher and lower molecular mass forms were observed. The amount of high-molecular-mass forms increased by time and was enhanced in the presence of Ub and clasto-lactacystin beta-lactone. In pulse-chase experiments the amount of full-length hTH1 decreased by first-order kinetics with a half-time of 7.4 h and 2.1 h in the absence and presence of an ATP-regenerating system, respectively. The ATP-dependent degradation was inhibited by clasto-lactacystin beta-lactone. Our findings support the conclusion that hTH1 is ubiquitinated and at least partially degraded by the proteasomes in the reticulocyte lysate system. Finally, it is shown that the integral TH of the bovine adrenal chromaffin granule membrane (bTHmem) is ubiquitinated, most likely monoubiquitinated. Additional Ub-conjugates of this membrane, detected by Western blot analysis, have not yet been identified.

Interaction of phosphorylated tyrosine hydroxylase with 14-3-3 proteins: evidence for a phosphoserine 40-dependent association.

Tyrosine hydroxylase (TH) has been reported to require binding of 14-3-3 proteins for optimal activation by phosphorylation. We examined the effects of phosphorylation at Ser19, Ser31 and Ser40 of bovine TH and human TH isoforms on their binding to the 14-3-3 proteins BMH1/BMH2, as well as 14-3-3 zeta and a mixture of sheep brain 14-3-3 proteins. Phosphorylation of Ser31 did not result in 14-3-3 binding, however, phosphorylation of TH on Ser40 increased its affinity towards the yeast 14-3-3 isoforms BMH1/BMH2 and sheep brain 14-3-3, but not for 14-3-3 zeta. On phosphorylation of both Ser19 and Ser40, binding to the 14-3-3 zeta isoform also occurred, and the binding affinity to BMH1 and sheep brain 14-3-3 increased. Both phosphoserine-specific antibodies directed against the 10 amino acids surrounding Ser19 or Ser40 of TH, and the phosphorylated peptides themselves, inhibited the association between phosphorylated TH and 14-3-3 proteins. This was also found when heparin was added, or after proteolytic removal of the N-terminal 37 amino acids of Ser40-phosphorylated TH. Binding of BMH1 to phosphorylated TH decreased the rate of dephosphorylation by protein phosphatase 2A, but no significant change in enzymatic activity was observed in the presence of BMH1. These findings further support a role for 14-3-3 proteins in the regulation of catecholamine biosynthesis and demonstrate isoform specificity for both TH and 14-3-3 proteins.

Cruciform-extruding regulatory element controls cell-specific activity of the tyrosine hydroxylase gene promoter.

Tyrosine hydroxylase (TH) is expressed specifically in catecholaminergic cells. We have identified a novel regulatory sequence in the upstream region of the bovine TH gene promoter formed by a dyad symmetry element (DSE1;-352/-307 bp). DSE1 supports TH promoter activity in TH-expressing bovine adrenal medulla chromaffin (BAMC) cells and inhibits promoter activity in non-expressing TE671 cells. DNase I footprinting of relaxed TH promoter DNA showed weak binding of nuclear BAMC cell proteins to a short sequence in the right DSE1 arm. In BAMC cells, deletion of the right arm markedly reduced the expression of luciferase from the TH promoter. However, deletion of the left DSE1 arm or its reversed orientation (RevL) also inactivated the TH promoter. In supercoiled TH promoter, DSE1 assumes a cruciform-like conformation i.e., it binds cruciform-specific 2D3 antibody, and S1 nuclease-cleavage and OsO4-modification assays have identified an imperfect cruciform extruded by the DSE1. DNase I footprinting of supercoiled plasmid showed that cruciformed DSE1 is targeted by nuclear proteins more efficiently than the linear duplex isomer and that the protected site encompasses the left arm and center of DSE1. Our results suggest that the disruption of intrastrand base-pairing preventing cruciform formation and protein binding to DSE1 is responsible for its inactivation in DSE1 mutants. DSE1 cruciform may act as a target site for activator (BAMC cells) and repressor (TE671) proteins. Its extrusion emerges as a novel mechanism that controls cell-specific promoter activity.

Isolation and Characterization of Ovine Tyrosine Hydroxylase mRNA

Tyrosine hydroxylase (TH) cDNA has been characterized in rodents and primates, but only a few studies have been developed in ungulates, except in cows. Because sheep is a species used for many physiological studies, it was of interest to clone TH cDNA in this species. Ovine TH cDNA was purified from a library of sheep adrenal glands. The entire cDNA was 1,721 bp long. It presented a higher percentage of similarity with bovine TH cDNA (93%) than with rodent cDNAs (75%). The deduced amino acid sequence was 490 amino acids long and had 96% similarity with the bovine amino acid sequence. The entire cDNA and different fragments obtained with endonuclease restriction enzymes were cloned in plasmid pUC 18 and were labeled with 35S-dATP to detect TH mRNA by in situ hybridization. Strong labelings were observed on adrenal medulla and on noradrenergic and dopaminergic neurons in the sheep but also in the cow and pig. This labeling matched completely TH immunohistochemical staining obtained on the same sections with anti-TH antibodies. Ovine TH cDNA is a useful tool to study the variations of TH mRNA levels in sheep catecholaminergic neurons.

Bovine tyrosine hydroxylase gene-promoter regions involved in basal and angiotensin II-stimulated expression in nontransformed adrenal medullary cells.

The tyrosine hydroxylase gene is expressed specifically in catecholaminergic cells, and its activity is regulated by afferent stimuli. To characterize molecular mechanisms underlying those regulations, we have constructed chimeric genes consisting of bovine tyrosine hydroxylase gene promoters (wild-type or deletion mutants) and a luciferase reporter gene. The basal expression of these genes and their regulation by angiotensin II were examined in cultured bovine adrenal medullary cells. Luciferase activity was normalized to the amount of transfected plasmid DNA. A pTHgoodLUC plasmid containing the -428/+21-bp fragment of the tyrosine hydroxylase gene promoter expressed luciferase activity at severalfold higher levels than the promoterless pOLUC plasmid. Deletion of the -194/-54-bp promoter fragment containing POU/Oct, SP1, and other putative regulatory elements increased luciferase expression fivefold. An additional deletion further upstream (-269/-194 bp), including a 12-O-tetradecanoylphorbol 13-acetate (TPA)-responsive element (TRE)-like site, reduced promoter activity. These results indicate the presence of negatively and positively acting regions in the bovine tyrosine hydroxylase gene promoter controlling basal promoter activity in adrenal medullary cells. Angiotensin II stimulated the expression of endogenous tyrosine hydroxylase gene and pTHgood-LUC approximately threefold without affecting the expression of pOLUC. A comparable threefold stimulation was observed following the deletion of the -194/-54-bp promoter region, despite the increase in basal promoter activity. Additional deletion of the -269/-194-bp promoter fragment reduced stimulation by angiotensin II to 1.5-fold. These results indicate that the angiotensin II receptor-responsive element is located in the -269/-194-bp promoter region containing the TRE-like site. Additional angiotensin II-responsive site(s) may be present outside this region. Gel mobility shift assays demonstrated constitutive and angiotensin II-induced protein binding to the tyrosine hydroxylase gene promoter. Some DNA-protein complexes were displaced with c-Fos antibodies. The results suggest that c-Fos-related antigens support basal promoter activity and mediate activation of tyrosine hydroxylase by angiotensin II receptor.

Regulation of tyrosine hydroxylase gene expression in depolarized non-transformed bovine adrenal medullary cells: second messenger systems and promoter mechanisms

Activation of the tyrosine hydroxylase (TH) gene in the adrenal medulla during stress is mediated by trans-synaptic mechanisms and may involve cholinergic receptors. Stimulation of nicotinic receptors in adrenal medullary cells induces cell depolarization, influx of Ca 2+ ions and increases levels of cAMP. We have shown that both cAMP and membrane depolarization produce an increase in the expression of the TH gene in cultured bovine adrenal medullary cells (BAMC). Others have proposed that transcriptional activation of the TH gene by cAMP is mediated through the sequence homologous to a cAMP responsive element (CRE) located in the proximal region of the TH gene promoter. In the present study we have examined the mechanisms by which membrane depolarization increases the TH gene activity. Treatment of serum-free BAMC cultures with the depolarizing agent, veratridine, increased the extracellular concentration of catecholamines, Met 5-enkephalin, and the relative abundance of TH mRNA. Veratridine treatment also increased the levels of mRNAs for the catecholamine biosynthetic enzyme phenylethanolamine N-methyltransferase (PNMT), and proenkephalin A (PEK). Treatment for longer than 3 h was required to increase TH mRNA levels. By contrast, our previous studies indicated that cAMP stimulation for 2 h produces a maximal increase in TH mRNA levels in BAMC. The effects of veratridine and forskolin on TH mRNA levels were additive, further indicating that depolarization and cAMP activate TH gene expression via different pathways. Calmidazolium, an antagonist of calmodulin, had no effect on the veratridine-induced increase in TH mRNA levels. Similarly sphingosine treatment or preincubation with PMA, which reduce protein kinase C (PKC) activity and attenuate the induction of TH mRNA by PMA or the hormone, angiotensin II, did not affect the induction by veratridine. To identify promoter mechanisms of TH gene activation in depolarized cells we transfected BAMC with a plasmid pTHgoodLuc and treated with veratridine for 24 h. pTHgoodLUC contains a luciferase reporter gene linked to a −428 +21 bp fragment of the bovine TH gene promoter (relative to the transcription start site). Veratridine increased the expression of luciferase from the TH promoter 2.5-fold. Deletion of the −194 −54 bp promoter region containing SP-1 and POU/Oct sites reduced veratridine stimulation by 40%. Additional deletion of the −269 to −190 bp promoter segment, including an AP-1 element, further reduced veratridine stimulation to a statistically non-significant level. In conclusion, activation of TH gene expression upon depolarization is not mediated by calmodulin and PKC. Promoter sequences involved in this activation are located upstream from the CRE. Depolarization may activate TH gene transcription by acting on more than one regulatory region.

A 5′-flanking region of the bovine tyrosine hydroxylase gene is involved in cell-specific expression, activation of gene transcription by phorbol ester, and transactivation by c-Fos and c-Jun

Tyrosine hydroxylase (TH) is expressed specifically in catecholaminergic cells and is transcriptionally activated via a protein kinase C (PKC)-dependent pathway. To identify regulatory regions of the TH gene, transfected neural crest-derived catecholaminergic cells [human neuroblastoma SH-SY5Y and bovine adrenal medullary (BAM) cells] or glia-derived SF-763 cells were used to analyze expression of a chloramphenicol acetyltransferase (CAT) reporter gene under the control of 5′-flanking sequences of the bovine TH gene. Plasmid pTH(−245/+21)CAT was constructed by fusing the −245 to +21 region of the TH gene to CAT sequences in the promoterless pCAT basic plasmid. Cells transfected with pTH(−245/+21)CAT expressed CAT activity at levels 8- to 100-fold higher than those of cells transfected with pCAT basic. In SH-SY5Y or BAM cells, pTH(−245/+21)CAT supported the expression of CAT at levels similar to or higher than that supported by the tissue nonspecific viral SV40 promoter/enhancer. In glia-like cells, CAT expression from pTH(−245/+2 1)CAT was about 13-fold lower than that under the control of the SV40 promoter. Incubation of neuroblastoma cells with the active phorbol ester, phorbol 12-myristate 13-acetate (PMA), increased expression of CAT from pTH(−245/+2 1)CAT over 6-fold and was accompanied by induction of c-fos and c-jun mRNAs and proteins. The regulation of TH promoter function by c-Fos/c-Jun was corroborated by transactivation of the TH promoter in SH-SY5Y cells engineered to overexpress exogenous c-Fos and c-Jun. TH gene sequences essential for c-Fos/c-Jun transactivation were located in the −245 to −52 region, upstream from a CRE-like element. Gel mobility assays demonstrated binding of c-Fos-antigen(s) to the region of the TH promoter that supports activation by PMA and c-Fos/c-Jun. Temporal patterns of nicotinic agonist-stimulated induction of c-fos and TH mRNA are also consistent with a role for c-Fos in TH gene transcription. Our results demonstrate that the 5′-flanking region of the bovine TH gene operates as an authentic promoter capable (i) of directing cell-specific expression of a bacterial CAT gene and (ii) of conferring responsivity to PKC-stimulation. The results also suggest that the nuclear proteins c-Fos and c-Jun contribute to PKC pathway-mediated activation of the TH gene via direct interaction with the TH gene promoter. The activation of TH gene expression by PKC/c-Fos/c-Jun may serve as an additional or alternative mechanism to activation by the cAMP-CRE pathway.

Cold-induced alterations in the binding of adrenomedullary nuclear proteins to the promoter region of the tyrosine hydroxylase gene.

It is well documented that cold stress induces a rapid trans-synaptically mediated increase in the relative abundance of rat adrenomedullary tyrosine hydroxylase (TH) mRNA. To investigate the transcriptional mechanisms regulating the cold stress response, we have employed a gel mobility shift assay, using DNA fragments prepared from the proximal 5' flanking region of the bovine TH gene as a heterologous molecular probe. In pilot studies, this region of the bovine TH promoter (nucleotides -246 to +21) was fused to the bacterial reporter gene, chloramphenicol acetyltransferase, and the chimeric construct transfected into human neuroblastoma SK-N-BE(2)-C, hepatoma HepG2, and rat pheochromocytoma PC-12 cells. Results of this analysis indicate that the proximal 5' flanking region of the bovine TH gene contains sufficient information to drive transient reporter gene expression in both human and rat catecholaminergic clonal cell lines. The findings derived from the gel mobility shift studies demonstrate that cold exposure causes rapid and selective alterations in the binding of adrenomedullary nuclear proteins to the proximal 5' flanking region of the TH gene. The most striking cold stress-induced alteration in DNA/nucleoprotein binding occurs in a region of the TH promoter (nucleotides -246 to -189) which contains an element bearing marked sequence similarity to an AP1 binding site and is highly conserved among animal species. This alteration occurs within 1 hr of cold exposure and persists for up to 48 hr after the onset of stress. The results of adrenal denervation experiments indicate that the cold-induced change in DNA/nucleoprotein binding is neurally mediated, requiring intact sympathetic innervation of the gland.(ABSTRACT TRUNCATED AT 250 WORDS)

The amino acid substrate of bovine tyrosine hydroxylase.

Tyrosine hydroxylase catalyzes the tetrahydropterin-dependent hydroxylation of tyrosine to form 3,4-dihydroxyphenylalanine. Several nonphysiological aromatic amino acids have been examined as inhibitors and substrates for bovine adrenal tyrosine hydroxylase. The Ki values for para-substituted phenylalanines increase as the size of the substituent increases. For each A2 increase in surface area of the substituent, the free energy of binding becomes 50 cal more positive. Replacement of the phenyl ring with a pyridyl ring decreases the affinity about one order of magnitude. A number of these aromatic amino acids are also substrates for the enzyme. The KM values again increase in size with increasing size of the substituent, but the Vmax value is independent of the reactivity of the amino acid. The effect of size on binding is consistent with a tight interaction between the para position region of the substrate and the enzyme. The lack of a change in the Vmax value is consistent with the rate-limiting step in catalysis by bovine tyrosine hydroxylase being formation of the hydroxylating intermediate rather than hydroxylation of the amino acid. These results will be useful in designing mechanism-based inhibitors of catecholamine biosynthesis and establish that the mechanisms of rat and bovine tyrosine hydroxylase do not differ significantly.

Simple and accurate assay for tyrosine hydroxylase from bovine retina by high-performance liquid chromatography with fluorescence detection.

We present a simple and sensitive assay for tyrosine hydroxylase (TH) using high-performance liquid chromatography (HPLC) with a fluorescence detector which enabled us to study bovine retinal TH of a small quantity in crude retinal samples. The detection limit with this method was 1 pmol of dihydroxyphenylalanine (DOPA) enzymatically produced. Retinal TH exhibited the maximum activity at pH 6 to 6.3, and almost none at physiological pH of above 7, a tendency similar to that reported for striatal TH.

Neural and hormonal regulation of the tyrosine hydroxylase gene in adrenal medullary cells: Participation of c-fos and AP1 factors

Previous studies identified nicotine and angiotensin receptors as major trans-synaptic and hormonal inputs controlling activities and mRNA levels of the catecholamine biosynthetic enzymes in adrenal medullary (AM) cells. The purpose of this study was to further explore the molecular mechanisms underlying these modes of regulations. Incubation of cultured bovine AM cells with nicotine or a stable analog of angiotensin II, sar1-angiotensin II, increased synthesis of tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) mRNAs (2- to 3-fold), suggesting transcriptional activation of these genes. No effects of nicotine or sar1-angiotensin II on TH and PNMT mRNA levels could be demonstrated in cells treated with cycloheximide or puromycin, suggesting regulation by inducible proteins. However, translational inhibitors produced small increases in basal TH mRNA levels (1.5- to 1.9-fold), indicating that repressor-like mechanisms participate in the regulation of the TH gene. Sequence analysis of the bovine TH promoter (1.6 kb) revealed the presence of several putative regulatory elements including four nonidentical AP1-like sites, potential targets for c-Fos/c-Jun/AP1 regulation. Formation of sequence-specific complexes between nuclear proteins and five fragments of the TH promoter (200–400 nt) was demonstrated by gel mobility shift assay (one to three complexes per fragment). Binding of proteins to three promoter fragments containing single AP1-like elements was antagonized by an oligodeoxynucleotide-containing AP1 consensus sequence (oligo-AP1), but not by other unrelated DNA sequences. Oligo-AP1 inhibited the formation of one or two complexes with individual DNA fragments. These observations suggest formation of different AP1-like complexes on individual promoter fragments. c-Fos antibody inhibited the formation of three oligo-AP1-displaceable complexes. The formation of the majority of retarded bands, including all AP1-like complexes, was increased by incubation of cells with sar1-angiotensin II or nicotine. Western analysis demonstrated the presence of c-Fos (p54), c-Jun (p36–38), and several c-Fos- and c-Jun-related antigens in control and stimulated AM cells. Sar1-angiotensin II and nicotine increased steady-state levels of c-Fos (1.5- to 2-fold) and c-fos mRNA (10- to 14-fold) and the levels of several c-Fos-related antigens (FRA) (2- to 7-fold). Some of FRA showed differential regulation by angiotensin II and nicotinic receptors. The levels of p36–38 c-Jun and c-jun mRNA were increased by sar1-angiotensin II (2- to 5-fold) and were less affected by nicotine. In conclusion, our results suggest transcriptional regulation of the TH gene by angiotensin II and nicotinic receptors, through partially different nuclear pathways. The regulation may involve multiple target sites in the TH promoter, inducible proteins, AP1-like factors, c-Fos, and c-Fos-related antigens.

Isolation and structural characterization of the bovine tyrosine hydroxylase gene

A bovine tyrosine hydroxylase (TH) cDNA probe was used to screen a charon 30 genomic library. Screening of approximately 1 million recombinant phage resulted in the identification of one clone, lambda B1, containing the entire bovine TH gene. Results derived from restriction endonuclease mapping and sequence analysis reveal that the bovine gene contains 13 exons spanning approximately 7 kb of genomic DNA. Determination of the transcription initiation site indicates that the TH gene has a 5' untranslated region of 27 bp. A TATA-box sequence is located between positions-29 and -24 from the transcription initiation site and a cyclic AMP regulatory element (CRE) between-45 and -38. Although the TH gene appears to be glucocorticoid responsive in vitro, no regions bearing identity to the consensus sequence for the glucocorticoid regulatory element (GRE) were detected within approximately 1.5 kb of 5' flanking sequence. A cross-species comparison of the 5' flanking sequences of the bovine, rat, and human TH genes reveals strong sequence and positional conservation of seven sequence elements. An analysis of the nucleotide sequence within these elements reveals similarity to the consensus sequences reported for known cis-acting regulatory elements and transcription factor binding sites, suggesting that they may play a role in the regulation of TH gene expression.

Isolation and characterization of the gene for drosophila tyrosine hydroxylase

A Drosophila cDNA homologous to a rat tyrosine hydroxylase (TH) probe has been isolated and sequenced. The deduced amino acid sequence predicts a 57,861 dalton protein with almost 50% identity with rat TH. In vitro transcription of the cDNA followed by in vitro translation yields a single protein species of ∼58,000 daltons. The in vitro translation product, as well as a protein of the same molecular weight from wild-type Drosophila head protein extracts, is recognized by an antibody made against bovine TH. The presence of TH enzymatic activity in heads was demonstrated. In situ hybridization to polytene chromosomes localized the gene to 65B. The comapping of the mutant pale to this same region, as well as its phenotype, suggests that pale may be a TH mutation.

Predicted amino acid sequence of bovine tyrosine hydroxylase and its similarity to tyrosine hydroxylases from other species.

The previously obtained cDNAs coding for bovine tyrosine hydroxylase (TH) mRNA (mRNATH) were further analyzed, and the entire nucleotide sequence was determined. The mRNATH consists of 1,706 nucleotides with an open reading frame for 491 amino acids, which corresponds to a calculated molecular weight of 55,011. The predicted amino acid sequence of bovine TH is compared with that of rat TH and shows a similarity of 66% in the amino terminal (amino acids 1-157) and 91% in the carboxy terminal (amino acids 158-491) region of the TH protein molecule. The carboxy terminal region has been shown to make up the catalytic site of TH and, therefore, is conserved to a greater extent in different species than the amino terminal region, which has been shown to be mainly responsible for the regulation of the catalytic activity of TH. Three of the four serine residues (Ser 8, 19, and 40) that have been shown to be substrates for various protein kinases in rat TH are also present in bovine TH and are located near the amino terminal end of the molecule. The amino acids from position 60 to position 66 of rat TH are not present in bovine TH, resulting in the absence of a predicted hydrophobic region as compared with rat TH. This difference could result in an altered degree of regulation by posttranslational phosphorylation and also association to cell organelle membranes of bovine TH as compared with rat TH.

Isolation and nucleotide sequence of a cDNA clone encoding bovine adrenal tyrosine hydroxylase: comparative analysis of tyrosine hydroxylase gene products.

Investigations into the structure and mechanisms regulating the expression of the genes involved in catecholamine biosynthesis have led to the isolation of a cDNA coding for bovine adrenal tyrosine hydroxylase (TH). The 1,722 bp cDNA contains the complete coding sequence and 3' untranslated region of the TH mRNA. The nucleotide sequence of the cDNA and the deduced amino acid sequence were compared to those reported for rat and human TH. Bovine TH shares 85% and 84% amino acid sequence identity with that of rat and human TH, respectively. Alignment of the amino acid sequences of rat, bovine, and human TH reveals that 79% of the residues are identical in all three species, indicating a strong evolutionary conservation of enzyme structure. Moreover, three of the four putative phosphorylation sites located in the N-terminal region of TH are conserved in these animal species. There are, however, some interspecies differences in TH gene products. The 3' untranslated region of bovine TH mRNA is 56 and 97 nucleotides shorter than rat and human TH mRNA, respectively. Additionally, the bovine protein is 7 and 6 amino acids smaller than its rat and human homologues. All of the absent amino acid residues of bovine TH are missing from an alanine-rich region in the N-terminal portion of the rat and human proteins (amino acids 51-68). Comparison of the size of bovine and rat TH mRNA and protein by northern blot and immunoblot analyses yielded differences consistent with those predicted from the nucleotide sequence data.

Tyrosine hydroxylase-immunoreactive neurons of the hypothalamus: A light and electron microscopic study

The localization and morphology of neurons, processes, and neuronal groups in the rat hypothalamus containing tyrosine hydroxylase-like immunoreactivity were studied using an antiserum to bovine tyrosine hydroxylase. This antiserum was thoroughly characterized by precipitation of enzyme activity, immunoblotting, and precipitation of cell-free translation products; a single molecular weight band was recognized by the antiserum. Absorption of the antiserum with purified tyrosine hydroxylase abolished immunocytochemical staining, while addition of bovine dopamine β-hydroxylase had no effect on immunostaining. Immunoreactive cells were found throughout the hypothalamus. Significant numbers of cells were found in the arcuate, periventricular, dorsomedial hypothalamus/zona incerta, posterior hypothalamic regions (A11–A14), and paraventricular nucleus, as previously described, and in addition, in the preoptic area, adjacent to the anterior commissure, medial and lateral to the suprachiasmatic nucleus, dorsal to and in the supraoptic nucleus, at the lateral borders of the ventromedial nucleus, and in the dorsal and ventral lateral hypothalamus. None of the immunoreactive cell groups are totally separated from adjacent cell groups. Dendritic overlap occurs between any two adjacent groups. From cell counts of 30 μm coronal sections, we estimate the hypothalamus has about 12,000 cells based on raw counts, or 8000 immunoreactive cells after correction for possible split cells. Mean soma size varied considerably from one immunoreactive group to another. Cells in the caudal part of the dorsomedial hypothalamus/zona incerta region were the largest, with a mean diameter of 25 μm, while cells in the anterior commissural and posterior hypothalamic group were among the smallest, with mean diameters of 10 μm. The largest immunoreactive cells in the hypothalamus had volumes in excess of ten times greater than the smallest immunoreactive cells. Tyrosine hydroxylase immunoreactivity was found in dendrites in every region of the hypothalamus, sometimes extending hundreds of micrometers from the perikaryon of origin. Although adjacent cell groups were not distinctly separated, the dendritic arbors of the different cell groups differed greatly. Dendritic and somatic appendages were found on some cells, particularly in the paraventricular nucleus. Immunoreactive dendritic arbors were particularly large in cells seen on horizontal sections through the caudal dorsomedial hypothalamic group and through the anterior hypothalamus. Only slight dendritic trees were observed in the rostral dorsomedial hypothalamus/zona incerta region, and in the pericommissural group. Immunoreactive axons, containing dopamine, norepinephrine or epinephrine, were found in every area of the hypothalamus. Axon morphology, size, density and terminals varied from region to region. Axons of hypothalamic origin arose from both immunoreactive somata and primary dendrites. A large number of non-synapsing immunoreactive axons were found in the median eminence, and many were also found throughout the hypothalamus. Retrograde transport of substances injected in the neurohypophysis labeled cells in the most rostral part of the arcuate nucleus and periventricular area; some labeled cells also showed tyrosine hydroxylase immunoreactivity. Many of the immunoreactive boutons contained primarily clear vesicles, while others contained both clear and large dense-core vesicles. Immunoreactive axons made synaptic contact with unlabeled dendrites.