Cloning and characterization of a novel trinucleotide repeat- containing gene GARP from Euplotes octocarinatus

Abstract

The expansion of trinucleotide repeats in genome is related to the phthogenesis of several neurodegenerative diseases. A GARP (glutamic acid-rich protein) gene was isolated from the macronuclear plasmid mini library of Euplotes octocarinatus. A micronuclear version of the GARP gene was amplified by polymerase chain reaction. The macronuclear DNA molecule carrying the GARP gene is 460 bp long and shows the characteristics of macronuclear chromosomes of hypotrichous ciliates. One of the three cysteines is encoded by the opal codon TGA(88-90). The predicted open reading frame encodes a 112-amino acid polypeptide, with a predicted molecular mass of 13 kDa and an isoelectric point of 3.82. Micronuclear version of the GARP gene contains two internal eliminated sequences (IES), IES1 and IES2. IES1 is 41 bp long and is flanked by 5'-GA-3' direct repeats. IES2 is 41 bp long and flanked by 5'-TA-3' direct repeats. Transcriptional activity of GARP gene was confirmed by reverse transcription polymerase chain reaction (RT-PCR).