Y-chromosomal <i>VCY2</i> arose via transposition of the autosomal fragments.

<p>(A) The presence (black dots), absence (white dots) of Chr-Y green-amplicon homologous sequences and <i>VCY2</i> homologs in representative primate species. The assumed date for the amplicon transposition was indicated by the arrow at the species tree of the primates. Status unknown (gray dots). (B) The synteny analysis of the green-amplicon from human Chr-Y and autosomal fragments from 8q11 and 7q11. We marked those autosomal sequences that have high sequence similarities with Chr-Y <i>TTTY17</i> and <i>TTTY4</i> but not annotated as genes. NWM, New World monkey; OWM, Old World monkey; <i>P</i>, pseudogene; TRIM, tripartite motif containing; <i>TTTY</i>, testis-specific transcript, Y-linked; <i>ZNF</i>, zinc finger protein.</p

Expression evidence for human <i>VCY2</i> and <i>BEYLA</i> genes.

<p>The EST (expressed sequence tag) and other evidence for human <i>VCY2</i> and <i>BEYLA</i> expression was shown. The accession number for each EST clone was listed.</p><p>Hosa (<i>Homo sapiens</i>).</p><p>Expression evidence for human <i>VCY2</i> and <i>BEYLA</i> genes.</p

Non-protein-coding <i>VCY2</i> on the rhesus macaque Chr-Y male-specific region.

<p>(A) Homologous fragments of <i>VCY2</i> from three overlapping BAC clones (CH250–99F15, CH250–59H13, and CH250–249M17) were extracted from the rhesus macaque male-specific region of Chr-Y (MSY). The position of each BAC clone in the whole MSY region is marked. (B) A putative synteny block around the <i>VCY2</i> from rhesus macaque was reconstructed and compared with the synteny block of human <i>VCY2</i>. (C) Fragments homologous to <i>VCY2</i> were extracted from rhesus macaque Chr-8 and Chr-Y. Artificial translations of rhesus macaque <i>VCY2</i> exon 1 and exon 4 are shown below each row of the alignment and compared to the deduced protein sequence of the <i>VCY2</i> ORF above the row. Positions of start and stop codons are indicated. Disablers are indicated by red. <i>DAZ</i>, deleted in the azoospermia protein; Hosa, <i>Homo sapiens</i>; Mamu, <i>Macaca mulatta</i>.</p

Non-protein-coding autosomal sequences homologous to <i>VCY2</i> on marmoset Chr-2.

<p>(A) The synteny block comparison around human <i>Chr-7-BEYLA</i> and its homologous sequences from marmoset Chr-2. The arrows indicates the orientation of each genes. Fragments homologous to <i>VCY2</i> extracted from human were used to compare with the fragments from marmoset. Protein-coding exons were joined to obtain the putative autosomal <i>VCY2</i> ORF. (B) The deduced protein sequence for <i>VCY2</i> ORF was shown above the row. Positions of start codons are indicated. Disablers are indicated by red. Caja, <i>Callithrix jacchus</i>; Hosa, <i>Homo sapiens</i>.</p

Model of the origin of <i>de novo</i> protein-coding genes on Chr-Y.

<p>A putative <i>de novo</i> protein-coding gene (green) acquired the protein-coding ability through (A) the transposition or retroposition of non-protein-coding autosomal segments or (B) X-degenerate sequences from an ancestral proto-sex chromosome, while non-protein-coding homologs remained so on autosomes or Chr-X (red).</p

<i>VCY2</i> gene families in human and chimpanzee genomes.

<p><i>VCY2</i> from human (GRCh38) and chimpanzee (CHIMP2.1.4) genomes were extracted and their Ensembl IDs and chromsomal location were shown. ‘1’ or ‘-1’ indicate the forward and reverse strand on the chromosome respectively. Human <i>BPY2DP</i> (basic charge, Y-linked, 2D) is a pseudogene of <i>VCY2</i>.</p><p>^aAll protein-coding paralogs encode a 106-a.a. Protein.</p><p>^ΨPseudogenes.</p><p>Hosa, <i>Homo sapiens</i>; Patr, <i>Pan troglodytes</i>.</p><p><i>VCY2</i> gene families in human and chimpanzee genomes.</p

Non-protein-coding autosomal sequences homologous to <i>VCY2</i> on human Chr-7-<i>BEYLA</i> and autosomes of chimpanzees and rhesus macaques.

<p>The synteny block comparison around human <i>Chr-7-BEYLA</i> and its homologous sequences from chimpanzee and rhesus macaque. The protein-coding genes were shown, the arrows indicates the orientation of each gene. Fragments homologous to <i>VCY2</i> were extracted from human, chimpanzee Chr-7 and rhesus macaque Chr-3. Protein-coding exons were joined to obtain the putative autosomal <i>VCY2</i> ORF. (B) Artificial translations of Chr-7-<i>BEYLA</i> ORF(a) exon 1 and exon 2 are shown below each row of the alignment and compared to the deduced protein sequence for <i>VCY2</i> ORF above the row. Positions of start and stop codons are indicated. Disablers are indicated by red. Hosa, <i>Homo sapiens</i>; Mamu, <i>macaca mulatta</i>; Patr, <i>Pan troglodytes</i>; <i>ZNF</i>: Zinc finger protein.</p

Reconstruction of the evolutionary history of <i>VCY2</i>.

<p>(A) The protein-coding ability of <i>VCY2</i> and its homologs in seven representative primate autosomes or Chr-Y in this study. The lineage relationship among these species was referred to the Ensembl database (<a href="http://www.Ensembl.org/" target="_blank">www.Ensembl.org/</a>). (B) Phylogenetic analysis of the <i>VCY2</i> gene and its homologs in primates. The phylogenetic tree was constructed based on the 84 nt around <i>VCY2</i> protein-coding exon 1 and from autosomal <i>BEYLA</i> using the Maxium likelihood method. Bootstrap values for 500 replicates are shown next to branches; branch lengths are indicated in the same units as the evolutionary distances used to construct the phylogenetic tree. Critical evolutionary events for <i>VCY2</i> (red and green arrows) and the origin of different clades (square brackets) are indicated. Caja, <i>Callithrix jacchus</i>; Gogo, <i>Gorilla gorilla</i>; Hosa, <i>Homo sapiens</i>; Mamu, <i>Macaca mulatta</i>; NWM, New World monkeys; OWM, Old World monkeys; Paan, <i>Papio anubis</i>; Patr, <i>Pan troglodytes</i>; <i>Popy</i>, <i>Pongo pygmaeus</i>.</p

Non-protein-coding autosomal sequences homologous to <i>VCY2</i> (<i>BEYLA</i>) from 8q11 in humans.

<p>(A) The <i>Hosa-BELYA</i> sequence homologous to <i>Hosa-VCY2</i> was extracted and protein-coding exons were joined based on the <i>Hosa-VCY2</i> ORF to obtain <i>Hosa-BEYLA</i> ORF(a) and (b). (B) Map of the positions of <i>Hosa-VCY2</i> protein-coding exons (black bars) and putative exons from autosomal homologs (white bars); 1 cm in the diagram = 2 kb in the genomic sequence. Artificial translations of <i>Hosa-BEYLA</i> ORF(a) and (b) for each exon are shown below each row of the alignment and compared to the deduced protein sequence of the <i>Hosa-VCY2</i> ORF above the row. Start and stop codons are indicated. The asterisk (*) denotes the premature stop codon in the translated sequence. Disablers are indicated by red. Hosa, <i>Homo sapiens</i>.</p

The schematic of analysis pipeline.

<p>The orthologs of primate Chr-Y genes from the non-primate species were identified through a Basic Local Alignment Search Tool Protein-Protein (BLASTP) search against a non-primate non-redundant (nr) protein sequences database using an Expect (E)-value threshold of 10^-4 and a sequence identity greater than 35%. The genes without any possible homologs in non-primate species were considered to be primate-specific Chr-Y orphan genes. These candidates were subsequently scrutinized for any evidence of a protein-coding homolog in Chr-X or autosomes. Those genes would be excluded if there was a protein-coding gene in primate autosomes or Chr-X. The synteny comparison and sequence alignments of the candidate genes and its non-protein-coding homologs on Chr-X or autosomes would be used subsequently as the supporting information for <i>de novo</i> origin.</p