Igor Del Vecchio

Prion-like Doppel gene (PRND) in the goat: genomic structure, cDNA, and polymorphisms

The genomic structure of the caprine Doppel gene (PRND) was determined using the ovine sequence as a scaffold to generate PCR fragments that were aligned with a cDNA sequence obtained from testicular mRNA. The caprine gene contains two exons, 89 and >2291 bp long, separated by a 1689-bp intron. Two mRNA isoforms of 3.2 and 4.8 kb were identified in the testis, as well as the exact transcription start site by fluorescently labeled oligonucleotide extension (FLOE). Like in sheep and cattle, the open reading frame (ORF) (537 bp) lies within exon 2 and is very much conserved in sheep (99.3%) and cattle (97%). The intronic sequence is also highly conserved (95.3%) compared with sheep, with the only exception of a 47-bp insertion. The PRND ORF was sequenced in 47 healthy and 17 TSE-affected goats of the Italian Ionica breed. Seven nucleotide positions showed variation: T28C, C65T, A151G, G286A, C385G, T451C, and T528C. Five were commonly represented polymorphisms: T28C, T451C, and T528C are silent mutations at codons L10, L151, and I176, respectively, while A151G and C385G determine a T51A and L129V amino acid change, respectively. The two remaining variants, C65T and G286A, were rare, leading to the amino acid substitutions S22F and E96K, respectively. None of the polymorphisms was significantly relatable to the TSE status, and the same result was obtained by the analysis of the combined haplotypes at the five major polymorphic sites, namely, T28C, C65T, A151G, G286A, and C385G.

Isolation and molecular characterization of rasfadin, a novel gene in the vicinity of the bovine prion gene.

Abstract. A novel gene, rasfadin (RASSF2) was identified close to the bovine prion gene, and its genomic structure was derived with a combination of exon trapping and RACE. The gene covers at least 28 kb and maps to the same chromosomal region as the prion gene in cattle, sheep, and human. The RASSF2 ORF is composed of 987 base pairs divided into nine exons and shows a high nucleotide (88%) and amino acid similarity (95%) with a previously described human cDNA, KIAA0168. The bovine 3′UTR region is significantly shorter than the human counterpart, but shares with it two highly conserved nucleotide blocks. The expression of the gene was investigated in brain, liver, and spleen. Alternative splicing yields a shorter product in the liver composed of only four exons. Computer analysis showed a highly significant similarity of the rasfadin protein with the Ras association (RalGDS/AF-6) domain family 2 and with the afadin family, respectively, for the longer brain/spleen and the shorter liver variants.

The doppel gene biology: a scientific journey from brain to testis, and return

Doppel is a newly recognized prion-like molecule encoded by a novel gene locus, PRND, located on the same chromosomal region of the prion (PRNP) coding gene. Doppel was considered a paralogue and the first member of the prion-gene family, possibly originated through an ancestral gene duplication event. Prion and doppel have different expression patterns, suggesting that the gene products exhibit different biological functions. Actually, doppel is not involved in the aetiology of the Transmissible Spongiform Encephalopathies (TSEs) or “prion diseases” and is highly expressed only within the testicular tissue, suggesting an important physiological role in the process of spermatogenesis. The restricted spatial and temporal expression profile of doppel has suggested its investigation within particular pathological contexts, such as cancers, showing that it might represent a novel and attractive diagnostic molecular marker and that might provide insights into the regulatory pathways of tumor-cell transformation.