Kazuto Kobayashi

Isolation of a novel cDNA clone for human tyrosine hydroxylase: Alternative RNA splicing produces four kinds of mRNA from a single gene

Human tyrosine hydroxylase (TH) cDNA was isolated by molecular cloning. Lambda gt 11 cDNA library constructed from human pheochromocytoma was screened with a synthetic 23-mer oligonucleotide complementary to rat TH mRNA. We found a novel type of cDNA clone whose N-terminal sequence is similar to but clearly distinct from each of the three types (type 1, 2 and 3) of TH cDNA reported by Grima et al. [Nature (1987) 326, 707-711]. It contains both the 12-bp insert characteristic of type 2 cDNA and the 81-bp sequence of type 3. This novel cDNA clone was designated as type 4. Southern blot analysis of human genomic DNA indicated that TH is encoded by a single gene. This suggests that the four different forms of TH mRNA are produced by alternative RNA splicing from a single primary transcript.

Tissue-specific and high-level expression of the human tyrosine hydroxylase gene in transgenic mice

Transgenic mice carrying multiple copies of the human tyrosine hydroxylase (TH) gene have been produced. The transgenes were transcribed correctly and expressed specifically in brain and adrenal gland. The level of human TH mRNA in brain was about 50-fold higher than that of endogenous mouse TH mRNA. In situ hybridization demonstrated an enormous region-specific expression of the transgene in substantia nigra and ventral tegmental area. TH immunoreactivity in these regions, though not comparable to the increment of the mRNA, was definitely increased in transgenic mice. This observation was also supported by Western blot analysis and TH activity measurements. However, catecholamine levels in transgenics were not significantly different from those in nontransgenics. These results suggest unknown regulatory mechanisms for human TH gene expression and for the catecholamine levels in transgenic mice.

Structure of the mouse tyrosine hydroxylase gene

The mouse tyrosine hydroxylase (TH) gene was isolated from a genomic library by cross-hybridization with human TH cDNA probe. Nucleotide sequence analysis of two overlapping genomic clones showed that this gene is split into 13 exons distributed about 7.5 kb in length. The transcription initiation site was determined by primer extension analysis with mouse adrenal gland poly(A)+RNA. The structure of the mouse TH gene was similar to that of the human TH gene, but it contained neither the alternative splice donor site around the 3-end of the first exon nor an independent exon corresponding to the second exon of the human TH gene. There were the canonical TATA and GC boxes, cyclic AMP responsive element (CRE), and AP1 binding site in the 5-flanking region of the mouse TH gene.

Expression of four types of human tyrosine hydroxylase in COS cells

Alternative splicing from a single gene produces four kinds of human tyrosine hydroxylase (types 1–4) which have structural diversity only in the N‐terminal region. We attempted expression of the type 1–4 enzymes in COS cells and performed kinetic analyses. All had enzymatic activities. The K m values of the four types for L‐tyrosine and 6‐methyl‐5,6,7,8‐tetrahydropteridine were similar, although their relative homospecific activities were clearly different. The type 1 enzyme displayed the highest activity.

Expression of two forms of human dopamine-β-hydroxylase in COS cells

Abstract We previously reported four different cDNA clones encoding human dopamine-β-hydroxylase (Kobayashi et al., Nucl. Acids Res., 17 (1989) 1089–1102). These clones were different in a 3′ untranslated region (types A and B) and/or in 6 nucleotides in mRNAs. The difference at nucleotide 910 caused an amino acid change between Ala (A) and Ser (S) at amino acid residue 304 (DBH/A and DBH/S). We succeeded in expressing both of DBH/A and DBH/S of type A cDNAs in COS cells. Both of the expressed proteins showed enzyme activities and immunoreactivities. The two proteins had similar kinetic constants, but had different homospecific activities (activities per enzyme protein); the homospecific activity of human DBH/S was low, approximately one thirteenth that of human DBH/A.

Putative ammo-terminal presequence for β-subunit of plant mitochondrial F1ATPase deduced from the amino-terminal sequence of the mature subunit

The α‐ and β‐subunits of sweet potato mitochondrial F1ATPase were purified from the F1 complex by gel filtration and ion‐exchange high‐performance liquid chromatography. Isoelectric focussing and N‐terminal amino acid sequencing indicated that the purified β‐subunit contains at least two polypeptides similar to each other. The N‐terminal 18 amino acid sequence of the β‐subunit showed homology to the amino acid sequence of the tobacco mitochondrial F1ATPase β‐subunit precursor deduced from the nucleotide sequence [(1985) EMBO J. 4, 2159‐2165] between residues 56 and 73, suggesting that the N‐terminal 55 amino acids of the tobacco precursor constitute the presequence required for mitochondrial targetting.

Chapter 7 – Tyrosine Hydroxylase

Publisher Summary nThis chapter explains how tyrosine hydroxylase (TH) catalyzes the conversion of L-tyrosine to 3,4- dihydroxyphenylalanine (L-DOPA), which is the initial and rate-limiting step in the biosynthetic pathway of catecholamines including dopamine, noradrenaline, and adrenalin. Human genetic researchers have found mutations that lead to TH deficiency, showing an infantile onset, progressive hypokinetic-rigidity with dystonia or a complex encephalopathy with neonatal onset. TH deficiency is an autosomal recessive disorder associated with the mutations in the TH gene. The clinical phenotype of the disease shows an infantile onset, progressive L-DOPA-responsive dystonia or a progressive encephalopathy with L-DOPA-nonrespon. Parkinsons disease is an age-related neurodegenerative disease caused by the progressive loss of dopamine neurons in the ventral midbrain and the consequent reduction of the dopamine level. The standard procedure to treat the disease is pharmacotherapy by oral administration of L-DOPA, but many patients gradually develop L-DOPA-induced dyskinesia and motor fluctuation. Gene therapy trials offer alternative, complementary approaches for clinical application of Parkinsons disease.

Structure and Expression of Genes Encoding Higher Plant Mitochondrial F1F0-ATPase Subunits

In higher plants, both mitochondria and chloroplasts have their own F1F0-ATPase, and the role of these two complexes in the supply of cellular ATP varies greatly among differentiated plant tissues. Plant mitochondrial F1 either consist of five subunits like those from the other organisms, or contain an additional sixth subunit depending on plant species, and these F1 subunits are distinct from subunits of the chloroplast CF1 of the same plant at least in several plant species examined. Unlike fungal and mammalian F1, where all the subunits are encoded by nuclear genes, the α-subunit of plant F1 is encoded by the mitochondrial genome and all the other subunits seem to be encoded by the nuclear genome. Detailed studies on plant mitochondrial F1 subunits and their genes, and their comparison with chloroplastic counterparts, are expected to provide valuable informations on structure, biosynthesis, regulation and evolution of two F1 complexes in plant cells.

Synthesis of the Nuclear DNA-Encoded Subunits of Higher Plant Cytochrome C Oxidase and F1ATPase

Active biogenesis of mitochondria occurs in higher plant cells without accompanying cell division in response to changes in physiological and environmental conditions. For instance, it is well known that a marked increase in respiratory activity in storage organs during seed germination is brought about by active mitochondrial biogenesis. As another example, reports from our laboratory have demonstrated that mitochondrial biogenesis takes place in sweet potato root tissue when the tissue is sliced and incubated under moist conditions at room temperature, namely wounded, or is infected with pathogens(1,2). In the former case, mitochondrial biogenesis is programmed during ontogenesis of individual plant, whereas in the latter, it is induced in response to stresses. We are interested in the question how mitochondrial biogenesis is programmed and induced in non-dividing cells of higher plants. At the present status of our knowledge, however, it seems to be very hard to make an approach toward the molecular mechanisms of the programming and induction of mitochondrial biogenesis. We should have much information about the mechanism of mitochondrial biogenesis itself in higher plant cells before we shall try to study the molecular mechanisms of the programming and induction of mitochondrial biogenesis.