Sequence analysis of hypothalamic parathyroid hormone messenger ribonucleic acid.
PTH-like peptides and messenger RNA (mRNA) have recently been detected in neural tissues, but it is uncertain whether this reflects the transcription of the PTH gene or that of a closely related gene. This possibility has, therefore, been investigated. PTH-like complementary DNA (cDNA) moieties of predicted size were readily generated from reverse transcribed brain (hypothalamic and extrahypothalamic tissue) and pituitary RNA, using polymerase chain reaction (PCR) with three sets of overlapping oligonucleotide primers designed to amplify PTH cDNA fragments of 285, 372, and 459 base pairs (bp). PCR reamplification of the largest hypothalamic moiety with an internal set of primers also generated a cDNA fragment of the predicted size (372 bp). Restriction endonuclease digestion with BstNI cleaved the largest hypothalamic cDNA moieties into smaller fragments of 217 and 242 bp, identical to the cleavage of parathyroidal PTH cDNA. Rapid amplification of cDNA ends of the 3'-flanking cDNA sequences also produced hypothalamic and extra-hypothalamic cDNA moieties identical in size (499 bp) to parathyroidal PTH cDNA. Southern analysis of these PCR and rapid amplification of cDNA end cDNA fragments further indicated homology with PTH cDNA. This homology was subsequently confirmed by nucleotide sequencing, which demonstrated complete homology between the neural and parathyroidal cDNA fragments. This homology extended over 673 bp (spanning nucleotides 31-709 of PTH cDNA), encompassing 95% of the entire parathyroidal PTH cDNA. The mRNA for this gene, determined by Northern blotting with a riboprobe for PTH mRNA, was of identical size to the parathyroidal PTH, but its abundance in brain was less than 0.01% of that expressed in the parathyroid glands. This transcript was not, however, detected in liver. The translation of this moiety in hypothalamic tissues was indicated by the presence of a protein in the rat hypothalamus that was immunoreactive with PTH-(1-84) antiserum and of comparable size to that in parathyroidal tissue. The abundance of this protein in hypothalamic tissue was approximately 0.25% of that in the parathyroid glands, suggesting tissue-specific differences in its rate of synthesis, processing, or degradation. These results, therefore, demonstrate that the brain is an extraparathyroidal site of PTH gene expression and suggest autocrine or paracrine roles for PTH in neural function.
Nutley
M T
MT
Department of Physiology, University of Alberta, Edmonton, Canada.
Parimi
S A
SA
Harvey
S
S
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D015153:Blotting, Western; D018076:DNA, Complementary / Q000737:chemistry; D007031:Hypothalamus / Q000378:metabolism*; D008297:Male; D008969:Molecular Sequence Data; D010281:Parathyroid Hormone / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000737:chemistry*; D051381:Rats; D017207:Rats, Sprague-Dawley
D018076:DNA, Complementary; D010281:Parathyroid Hormone; D012333:RNA, Messenger