Functional copies of the Mst77F gene on the Y chromosome of Drosophila melanogaster

Abstract

The Y chromosome of Drosophila melanogaster has <20 protein-coding genes. These genes originated from the duplication of autosomal genes and have male-related functions. In 1993, Russell and Kaiser found three Y-linked pseudogenes of the Mst77F gene, which is a testis-expressed autosomal gene that is essential for male fertility. We did a thorough search using experimental and computational methods and found 18 Y-linked copies of this gene (named Mst77Y-1-Mst77Y-18). Ten Mst77Y genes encode defective proteins and the other eight are potentially functional. These eight genes produce approximately 20% of the functional Mst77F-like mRNA, and molecular evolutionary analysis shows that they evolved under purifying selection. Hence several Mst77Y genes have all the features of functional genes. Mst77Y genes are present only in D. melanogaster, and phylogenetic analysis confirmed that the duplication is a recent event. The identification of functional Mst77Y genes reinforces the previous finding that gene gains play a prominent role in the evolution of the Drosophila Y chromosome.

Figure 1.—

Mst77Y genes identified by de novo sequencing. Transcribed genes are marked with arrows; genes in which no transcript was detected are represented by boxes; open arrows and boxes, genes with intact ORFs (“potentially functional”); shaded arrows and boxes, genes with disrupting mutations (“nonfunctional”). Neighbor-joining tree of aligned nucleotides of the coding sequence is shown; branch lengths and the outgroups are shown in Figure S2 A. Only bootstrap values >50% are shown.

Figure 2.—

Quantification of the number of Mst77Y genes and their mRNA expression through XbaI digestion. PCR amplification was carried with primers that amplify both Mst77F and Mst77Y genes, using as templates male genomic DNA (left) and testis cDNA (right). The products were digested with the XbaI restriction enzyme, which cuts the Mst77Y sequences, but not Mst77F. UD, undigested PCR product; D1–D3, three independent PCR/digestion replicas; positive control, digestion of PCR from a cloned Mst77Y gene. The relative intensity of the 110-bp band (compared to the 179-bp band) estimates the number of Mst77Y genes or their mRNA expression (see text and Table 2).

Figure 3.—

Quantification of the number of Mst77Y genes through computational analysis. The origin (Mst77F or Mst77Y) of each trace of the genome project that has similarity to these genes was identified by its best BlastN match. We quantified this by annotating for each trace the number of aligned bases (identity × alignment length) to Mst77F and to one of the Mst77Y genes (Mst77-ψ1) and subtracting the former from the latter (“Alignment difference”). Positive values indicate that the trace came from a Mst77Y gene, and negative values indicate Mst77F origin.

Figure 4.—

Molecular evolution of Mst77Y genes. The four tests of neutral evolution of the Mst77F genes were based on d_N/d_S ratios (ω), which were constrained to 1 (“ω = 1,” dashed lines) or estimated from the data (“ω = free,” solid and shaded lines, one for each independently estimated ω). The estimated ω is shown in parentheses (Table S2 and Table S3 show the values of d_N and d_S). The result of the LRT and the biological interpretation are shown in column 4. “Ypf,” potentially functional Mst77Y genes (i.e., with intact ORF); “Ynf,” nonfunctional Mst77Y genes (with disrupting mutations). The figure is a simplification; the actual tests include the outgroups shown in Figure S6 and Table S3 (ω was free in all these branches). Tests were performed with the HyPhy package (Kosakovksy Pond et al. 2005), using Files S3 and S4.