Uboldi Cristina, Del Vecchio Igor, Foti Maria Gabriella, Azzalin Alberto, Paulis Marianna, Raimondi Elena, Vaccari Gabriele, Agrimi Umberto, Di Guardo Giovanni, Comincini Sergio, Ferretti Luca
Dipartimento di Genetica e Microbiologia, Università di Pavia, via Ferrata 1, 27100, Pavia, Italy.
Mamm Genome. 2005 Dec;16(12):963-71. doi: 10.1007/s00335-005-0084-1. Epub 2005 Dec 8.
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.
利用绵羊序列作为支架来确定山羊多普蛋白基因（PRND）的基因组结构，以生成与从睾丸mRNA获得的cDNA序列比对的PCR片段。山羊基因包含两个外显子，长度分别为89 bp和大于2291 bp，由一个1689 bp的内含子隔开。在睾丸中鉴定出了3.2 kb和4.8 kb的两种mRNA亚型，以及通过荧光标记寡核苷酸延伸（FLOE）确定的精确转录起始位点。与绵羊和牛一样，开放阅读框（ORF）（537 bp）位于外显子2内，在绵羊（99.3%）和牛（97%）中高度保守。与绵羊相比，内含子序列也高度保守（95.3%），唯一的例外是有一个47 bp的插入。对意大利爱奥尼亚品种的47只健康山羊和17只感染传染性海绵状脑病（TSE）的山羊的PRND ORF进行了测序。七个核苷酸位置出现变异：T28C、C65T、A151G、G286A、C385G、T451C和T528C。其中五个是常见的多态性：T28C、T451C和T528C分别是密码子L10、L151和I176处的沉默突变，而A151G和C385G分别导致T51A和L129V氨基酸变化。其余两个变异体C65T和G286A很少见，分别导致氨基酸替代S22F和E96K。没有一个多态性与TSE状态有显著相关性，对五个主要多态性位点（即T28C、C65T、A151G、G286A和C385G）的组合单倍型进行分析也得到了相同的结果。
