PG25, a pineal-specific cDNA, cloned by differential display PCR (DDPCR) and rapid amplification of cDNA ends (RACE).
Synthesis of melatonin in the mammalian pineal gland is regulated by the rhythmic expression of acetyl-CoA: serotonin N-acetyltransferase (SNAT) and other unknown factors. To screen for pineal-specific mRNAs potentially involved in melatonin synthesis and/or regulation, differential display PCR (DDPCR) was employed. We used 80 primer pairs and examined 40 bands of interest. One of the pineal specific clones (relative to brain and eye), PG25, was studied further. Hybridization histochemical and Northern analyses confirmed its tissue specificity. The size of the corresponding mRNA is 2.4 kb. A cDNA (2 kb) containing the coding region was obtained using a long-template PCR-based RACE technique. A data base search indicates that PG25 is highly homologous to a recently identified human lung endothelial cell-specific gene, ESM-1. Interestingly, not only the amino acid sequences but also the cDNA sequences, including the long 3' untranslated regions, are highly similar. This suggests that the conserved 3' untranslated region may carry information to regulate its own expression. Northern analysis revealed that PG25 is also expressed in the rat lung, but at a much lower (10%) level compared to the pineal. Finally, our work shows the feasibility of a fast, integrated PCR-based cloning method for obtaining long, potentially full-length cDNAs with restricted expression in anatomically complex regions of the brain. This protocol combining several existing methodologies is suitable for use with limited tissue sources and uses minimal amounts of isotopes.
Wang
X
X
Laboratory of Cellular and Molecular Regulation, NIMH, Bethesda, MD 20892, USA.
Brownstein
M J
MJ
Young
W S
WS
D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D005784:Gene Amplification; D006651:Histocytochemistry; D017403:In Situ Hybridization; D008297:Male; D008969:Molecular Sequence Data; D010870:Pineal Gland / Q000502:physiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D051381:Rats; D017207:Rats, Sprague-Dawley
D018076:DNA, Complementary