Molecular cloning and characterization of cDNA encoding tryptophan hydroxylase from rat central serotonergic neurons.
Tryptophan hydroxylase (TPH) from central serotonergic neurons in the dorsal raphe nucleus (DRN) and that from the endocrine pineal gland (PG) have been shown to exhibit difference biochemical characteristics. We further report here that the isoelectric point determined by chromatofocusing differs between TPH from the rat brainstem and PG. In addition, the levels of TPH mRNA are much greater in the PG than the DRN despite a higher enzymatic activity in the DRN. These data raise the question as to whether different forms of TPH may exist in the DRN and the PG. To address this question, we amplified TPH cDNAs by the polymerase chain reaction (PCR) using poly(A)+ RNA purified from both tissues. Several combinations of oligonucleotide primers encompassing different regions of the published coding sequence of rat pineal TPH were employed for this purpose. Subsequent analysis by gel electrophoresis and Southern blotting of PCR products indicated that DNA fragments of identical length were amplified from both sources. Furthermore, the nucleotide sequences of three independent subclones containing the putative full-length coding region of DRN TPH were determined and found to be identical to that of PG. In situ hybridization using the amplified cDNA as a probe demonstrated specific labeling within the DRN of the rat brain. These data support the hypothesis that tissue-specific differences in TPH characteristics result from differential post-translational events and clearly indicate that a TPH mRNA transcript identical in coding sequence to the PG form is expressed in the DRN.
Kim
K S
KS
Laboratory of Molecular Neurobiology, Cornell University Medical College, Burke Rehabilitation Center, White Plains, NY 10605.
Wessel
T C
TC
Stone
D M
DM
Carver
C H
CH
Joh
T H
TH
Park
D H
DH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D001921:Brain / Q000201:enzymology*; D001933:Brain Stem / Q000201:enzymology; D004247:DNA / Q000235:genetics*; D008969:Molecular Sequence Data; D009474:Neurons / Q000201:enzymology*; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D010870:Pineal Gland / Q000201:enzymology*; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D011903:Raphe Nuclei / Q000201:enzymology; D051381:Rats; D011919:Rats, Inbred Strains; D012701:Serotonin / Q000502:physiology*; D014365:Tryptophan Hydroxylase / Q000235:genetics*
D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D012701:Serotonin; D004247:DNA; D014365:Tryptophan Hydroxylase