Supplementary Material for: Molecular Characterization of the <i>DDX3Y</i> Gene and Its Homologs in Cattle

<i>DDX3Y</i> (also known as <i>DBY</i>) is a member of the DEAD box protein family, which is involved in ATP-dependent RNA unwinding, needed in a variety of cellular processes including splicing, ribosome biogenesis and RNA degradation. In the human, <i>DDX3Y </i>is located in the <i>AZFa</i> interval in the Y chromosome. Deletion of the <i>AZFa</i> region has been shown to disrupt spermatogenesis, causing subfertility and infertility in otherwise healthy men. Here, we report the characterization of the bovine (b) <i>DDX3Y</i> gene and its homologs <i>DDX3X </i>and <i>PL10</i>. We found 2 transcripts for the b<i>DDX3Y</i> (b<i>DDX3Y</i>-L and -S), which correspond to the long and short transcripts of the human <i>DDX3Y</i> and mouse <i>Ddx3y</i> gene. The 2 transcripts are identical except for a 3-bp (AGT) insertion at the position of nt 2025 and an expanded 3′UTR (nt 2155–2769) in b<i>DDX3Y</i>-L. The b<i>DDX3Y</i>-S encodes a peptide of 660 amino acids (aa), while the b<i>DDX3Y</i>-L encodes a peptide of 661 aa as the result of an additional serine (S) insertion at the position of aa 634. Both b<i>DDX3Y</i> isoforms contain the conserved DEAD-box motif. The b<i>DDX3Y </i>is composed of 17 exons. The homologous gene on the X chromosome, b<i>DDX3X</i>, is highly conserved to the Y-copy at mRNA (83%) and protein (88%) levels as well as in the genomic structure. The autosomal copy, b<i>PL10</i>, mapped on BTA15, is a processed pseudogene with a similarity of 88.1% to b<i>DDX3Y</i> and 93.7% to b<i>DDX3X</i> mRNA, suggesting that <i>PL10</i> is a retroposon of <i>DDX3X</i>. RT-PCR analyses showed that b<i>DDX3Y</i>-L, -S, b<i>DDX3X</i> and b<i>PL10</i> were all widely expressed with predominant expression in testis and brain. Testicular section in situ hybridization revealed that sense and anti-sense RNAs of b<i>DDX3Y</i>-L, -S, and b<i>DDX3X</i> were expressed in interstitial cells. These results together with the finding that the pseudogene b<i>PL10</i> is transcriptionally active in this study provide a basis for further investigating the <i>DDX3</i> gene function in spermatogenesis, male fertility and gene evolution in mammals.