Organization and evolution of the rat tyrosine hydroxylase gene

Brown, Elaine R.; Coker, George T.; O’Malley, Karen L.

doi:10.1021/bi00390a046

Cited by 77 publications

(46 citation statements)

References 30 publications

(24 reference statements)

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“…This vector expresses TH from the 5' long terminal repeat (LTR) and the selectable bacterial neomycinresistance gene (neo) from an internal Rous sarcoma virus (RSV) promoter (7). A 1688 base-pair (bp) BamHI/Sph I fragment from the plasmid pTH-54 (8)(9) containing the rat TH cDNA, a 1321-bp HindIII/Sma I fragment of neo from the plasmid pSV2neo (10), a 300-bp BamHI/HindIII fragment from the plasmid pUCRH containing a modified RSV promoter (J.-K. Yee, personal communication), and LTR sequences derived from the LPL2 vector (11) were ligated and cloned by standard methods (12). The polyadenylylation signal within the RSV promoter was changed from AATAAA to AGCAAA by site-directed mutagenesis to allow full-length transcripts from the 5' LTR promoter to the polyadenylylation site within the 3' LTR.…”

Section: Methodsmentioning

confidence: 99%

Grafting fibroblasts genetically modified to produce L-dopa in a rat model of Parkinson disease.

Wolff

Fisher

et al. 1989

Proc. Natl. Acad. Sci. U.S.A.

238

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Rat fibroblasts were infected with a retroviral vector containing the cDNA for rat tyrosine hydroxylase [TH; tyrosine 3-monooxygenase; L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2]. A THpositive clone was identified by biochemical assay and immunohistochemical staining. When supplemented in vitro with pterin cofactors required for TH activity, these cells produced L-dopa and released it into the cell culture medium. Uninfected control cells and fibroblasts infected with the TH vector were grafted separately to the caudate of rats with unilateral 6-hydroxydopamine lesions of the nigrostriatal pathway. Only grafts containing TH-expressing fibroblasts were found to reduce rotational asymmetry. These results have general implications for the application of gene therapy to human neurological disease and specific implications for Parkinson disease.A strategy for gene therapy, in which appropriate target cells are removed from the body, genetically modified in culture, and then implanted back into the body, has attracted considerable attention (1, 2). Recently, this strategy has been applied to the central nervous system by implanting genetically modified fibroblasts into the brain (3-5). The present study was undertaken to determine whether this approach could be used to reduce behavioral abnormalities in an animal model of human neurological disease.Parkinson disease may be an ideal candidate for treatment with this approach since the neurological abnormalities are known to result from degeneration of a specific class of cells, the dopaminergic neurons of the nigrostriatal pathway. The clinical signs of the disease can be ameliorated by replacement of L-dopa, the precursor of dopamine. Fibroblasts genetically modified to produce L-dopa might be capable of altering the phenotype of this disease after implantation into the brain. To investigate this possibility, a rat fibroblast cell line was infected in vitro with a retroviral vector containing the rat cDNA for the enzyme tyrosine hydroxylase [TH; tyrosine 3-monooxygenase; L-tyrosine, tetrahydrobiopteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2]. This enzyme, in the presence of the cofactor tetrahydropterin, catalyzes the conversion of tyrosine to L-dopa. Since tyrosine and tetrahydropterin are readily available to cells of the brain (6), TH-expressing fibroblasts should be capable of producing L-dopa when implanted intracerebrally. In this report, we demonstrate that genetically modified fibroblasts can produce and release L-dopa in vitro, and they can reduce behavioral abnormalities in an animal model of Parkinson disease. METHODSA retroviral vector containing the full-length rat TH cDNA (LThRNL) was derived from the Moloney murine leukemia virus (Fig. 1). This vector expresses TH from the 5' long terminal repeat (LTR) and the selectable bacterial neomycinresistance gene (neo) from an internal Rous sarcoma virus (RSV) promoter (7). A 1688 base-pair (bp) BamHI/Sph I fragment from the plasmid pTH-54 (8...

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Section: Methodsmentioning

confidence: 99%

Grafting fibroblasts genetically modified to produce L-dopa in a rat model of Parkinson disease.

Wolff

Fisher

et al. 1989

Proc. Natl. Acad. Sci. U.S.A.

238

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“…We fused upstream sequences from the TH promoter [2] to the 5′ end of a NF-H promoter [33]. The TH-NFH promoter supported expression for 1 month in either perirhinal or postrhinal cortices, 2 months in the hippocampus, and 6 months in the striatum, (the longest time points examined; helper virus-free system [50]).…”

Section: Introductionmentioning

confidence: 99%

Isolation of an enhancer from the rat tyrosine hydroxylase promoter that supports long-term, neuronal-specific expression from a neurofilament promoter, in a helper virus-free HSV-1 vector system

et al. 2007

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Direct gene transfer into neurons, using a virus vector, has been used to study neuronal physiology and learning, and has potential for supporting gene therapy treatments for specific neurological diseases. Many of these applications require high-level, long-term recombinant gene expression, in forebrain neurons. We previously showed that addition of upstream sequences from the rat tyrosine hydroxylase (TH) promoter to a neurofilament heavy gene (NF-H) promoter supports long-term expression in forebrain neurons, from helper virus-free Herpes Simplex Virus (HSV-1) vectors. This element in the TH promoter satisfied the definition of an enhancer; it displayed activity at a distance from the basal promoter, and in both orientations. This enhancer supported physiological studies that required long-term expression; a modified neurofilament promoter, containing an insulator upstream of the TH-NFH promoter, supported expression in ∼11,400 striatal neurons at 6 months after gene transfer, and expression for 7, 8, or 14 months, the longest times tested. In contrast, the NF-H promoter alone does not support long-term expression, indicating that the critical sequences are in the 6.3 kb fragment of the TH promoter. In this study, we performed a deletion analysis to identify the critical sequences in the TH promoter that support long-term expression. We localized these critical sequences to an ∼320 bp fragment, and two subfragments of ∼100 bp each. Vectors that contained each of these small fragments supported levels of long-term, neuronal-specific expression that were similar to the levels supported by a vector that contained the initial 6.3 kb fragment of the TH promoter. These small fragments of the TH promoter may benefit construction of vectors for physiological studies, and may support studies on the mechanism by which this enhancer supports long-term expression.

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“…Indeed, the three enzymes are 60% identical in their 330 C-terminal amino acids. Furthermore, comparison of the sequences of the rat tyrosine hydroxylase and human phenylalanine hydroxylase genes shows each consisting of I3 exons, with 10 of 12 intronlexon junctions conserved; the divergence is at the 5' end of the gene (DiLella et al, 1986;Brown et al, 1987).…”

mentioning

confidence: 99%

Expression and characterization of catalytic and regulatory domains of rat tyrosine hydroxylase

1993

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Phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase constitute a family of tetrahydropterin-dependent aromatic amino acid hydroxylases. Comparison of the amino acid sequences of these three proteins shows that the C-terminal two-thirds are homologous, while the N-terminal thirds are not. This is consistent with a model in which the C-terminal two-thirds constitute a conserved catalytic domain to which has been appended discrete regulatory domains. To test such a model, two mutant proteins have been constructed, expressed in Escherichia coli, purified, and characterized. One protein contains the first 158 amino acids of rat tyrosine hydroxylase. The second lacks the first 155 amino acid residues of this enzyme. The spectral properties of the two domains suggest that their three-dimensional structures are changed only slightly from intact tyrosine hydroxylase. The N-terminal domain mutant binds to heparin and is phosphorylated by CAMP-dependent protein kinase at the same rate as the holoenzyme but lacks any catalytic activity. The C-terminal domain mutant is fully active, with V,,, and K,,, values identical to the holoenzyme; these results establish that all of the catalytic residues of tyrosine hydroxylase are located in the C-terminal330 amino acids. The results with the two mutant proteins are consistent with these two segments of tyrosine hydroxylase being two separate domains, one regulatory and one catalytic.

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Organization and evolution of the rat tyrosine hydroxylase gene

Cited by 77 publications

References 30 publications

Grafting fibroblasts genetically modified to produce L-dopa in a rat model of Parkinson disease.

Grafting fibroblasts genetically modified to produce L-dopa in a rat model of Parkinson disease.

Isolation of an enhancer from the rat tyrosine hydroxylase promoter that supports long-term, neuronal-specific expression from a neurofilament promoter, in a helper virus-free HSV-1 vector system

Expression and characterization of catalytic and regulatory domains of rat tyrosine hydroxylase

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