Cloning of two mRNA variants of brain aromatase cytochrome P450 in rainbow trout (Oncorhynchus mykiss Walbaum).
This work describes the molecular cloning of the cDNA encoding the rainbow trout (Oncorhynchus mykiss Walbaum) brain cytochrome P450arom by means of reverse transcriptase and polymerase chain reaction (RT-PCR) and 5'- and 3'-rapid amplification of cDNA ends (RACE) analyses. The results obtained demonstrate that, as in other teleost fishes, the trout genome contains, besides the gene previously identified in the ovary, a second CYP19 gene (CYP19B) expressed at high level in the brain. Moreover, two P450aromB mRNAs, forms I and II, were found to be transcribed in trout. Form I (1816 sequenced nt) contains an open reading frame (ORF) of 1464b, a 5'-untranslated terminal region (UTR) of 124b and at least 228b in the 3'-UTR (incomplete, as the polyadenylation signal was not determined). Form II (1930 sequenced nt) contains an ORF of 1362b, a 5'-UTR of 340b and the same 3'-UTR as form I. Form II lacks the first 34 amino acids of form I, corresponding to the membrane-anchoring segment, whereas the sequence of the remaining coding region is almost the same in the two forms, resulting in proteins of 454 and 488 amino acids, respectively. Whether the two transcripts derive from the same gene by alternative splicing or are encoded by different CYP19B genes remains to be clarified. On Northern blot analyses with brain and ovary specific ORF probes and poly(A)(+)-enriched RNAs from trout ovary and brain, a transcript of about 2.6kb was identified in the ovary, as expected, whereas the full-length mRNA of brain P450arom is about 3.8kb. The brain form is expressed in the brain and gonads, whereas expression in peripheral tissues is limited mostly to the gills. The two trout CYP19 genes are not equivalent in tissue-specific expression, indicating the possibility of distinct promoters and regulatory mechanisms.
Dalla Valle
Luisa
L
Comparative Endocrinology Laboratory, Department of Biology, University of Padova, Via Uu Bassi 58/B, Italy. luisadv@mail.bio.unipd.it
Ramina
Annalisa
A
Vianello
Silvia
S
Belvedere
Paola
P
Colombo
Lorenzo
L
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001141:Aromatase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D001921:Brain / Q000201:enzymology*; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D005260:Female; D014644:Genetic Variation / Q000235:genetics*; D005880:Gills / Q000201:enzymology; D008969:Molecular Sequence Data; D017686:Oncorhynchus mykiss; D010053:Ovary / Q000201:enzymology; D010802:Phylogeny; D015347:RNA Probes; D012333:RNA, Messenger / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014018:Tissue Distribution
D017931:DNA Primers; D015347:RNA Probes; D012333:RNA, Messenger; D001141:Aromatase