A Novel Rat Tyrosine Hydroxylase mRNA Species Generated by Alternative Splicing

Abstract

Tyrosine hydroxylase (TH) catalyzes the first and rate-limiting step in the biosynthesis of catecholamines. Among the various mechanisms implicated in the regulation of TH activity, alternative splicing of TH primary transcript has been described as a characteristic of higher primates and Drosophila. We investigated whether there is such a regulatory mechanism in the rat. Reverse transcriptase-PCR experiments were performed with RNA from PCi 2 cells. A new TH mRNA species was evidenced, resulting from the use of an alternative donor site in exon 2. RNase protection assays and in situ hybridization experiments detected this mRNA species in the adrenal medulla but not in the main catecholaminergic nuclei of the CNS. The corresponding putative protein lacks 33 amino acids in the N-terminal regulatory domain. A recombinant protein was produced in E. co/i. Its in vitro specific activity was similar to that of the previously identified TH protein. Key Words: Rat-Tyrosine hydroxylase-Tyrosine hydroxylase mRNA species-Alternative splicing -Adrenal medulla-Tyrosine hydroxylase regulation.

Vasopressinergic neurons of the supraoptic nucleus in perinatal rats: reaction to osmotic stimulation and its regulation

“…The VP-41 sequence has no counterpart with oxytocin mRNA. The specificity of the probes VP-41 and TH-64 has been demonstrated previously (Trembleau et al 1988;Lanièce et al 1996). The probes were radiolabeled with [ 35 S], and in situ hybridization has been processed as described earlier (Marsais and Calas 1999).…”

“…After storing at -80°C, the cryostat sections were warmed at room temperature and rinsed in sterile PBS. Two oligonucleotide probes have been used for in situ hybridization: (1) VP-41, a 41-mer oligonucleotide probe complementary to the mRNA sequence coding for 115-128 amino acid residues of preproVP (Trembleau et al 1988); and b) TH-64, a 25-mer probe complementary to exon 2 of the TH mRNA sequence (Lanièce et al 1996). The VP-41 sequence has no counterpart with oxytocin mRNA.…”

A dynamic shift of VEGF isoforms with a transient and selective progesterone-induced expression of VEGF189regulates angiogenesis and vascular permeability in human uterus

“…The slides were hybridized overnight at 42°C with sense or antisense oligonucleotide probes specific for all VEGF transcripts (sequence corresponding to exon 4) or only V 189 transcripts (sequence corresponding to exon 6) (15); exon 4 sense probe, 5Ј-CTCACCAAGGCCAGCACATAGGAGAGATGA-3Ј; antisense probe, 5Ј-TCATCTCTCCTATGTGCTGGCCTTGGT-GAG-3Ј; exon 6 sense probe, 5Ј-GCAAGAAATCCCGGT-ATAAGTCCTGGAGCG-3Ј; antisense probe, 5Ј-CGCTC-CAGGACTTATACCGGGATTTCTTGC-3Ј; these probes were labeled with [ 35 S]ATP at the 3Ј end by using terminal deoxynucleotidyl transferase. After hybridization (30), the slides were exposed with photographic emulsion for 3-5 weeks.…”

Tissue‐specific developmental requirements of Drosophila tyrosine hydroxylase isoforms

“…In all species studied from human (Grima et al, 1985;O'Malley et al, 1987) to Drosophila (Neckameyer and Quinn, 1989;Birman et al, 1994), it has been found that TH is encoded by a single gene (Kumer and Vrana, 1996). Alternative splicing of the TH primary transcript has been demonstrated in human (Grima et al, 1987;Nagatsu and Ichinose, 1991;Dumas et al, 1996), monkey (Ichikawa et al, 1990), rat (Schussler et al, 1995;Laniece et al, 1996), and Drosophila (Birman et al, 1994). In all these species, the cassette exons that are alternatively spliced encode peptide modules in the amino terminal regulatory domain of the protein.…”