Acquisition and amplification of a testis-expressed autosomal gene, SSL, by the Drosophila Y chromosome

Abstract

The acquisition of autosomal fertility genes has been proposed to be an important process in human Y chromosome evolution. For example, the Y-linked fertility factor DAZ (Deleted in Azoospermia) appears to have arisen after the transposition and tandem amplification of the autosomal DAZH gene. The Drosophila melanogaster Y chromosome contains tandemly repeated Su(Ste) units that are thought to affect male fertility as suppressors of the homologous X-linked Stellate repeats. Here we report the detection of a testis-expressed autosomal gene, SSL [Su(Ste)-like], that appears to be an ancestor of the Y-linked Su(Ste) units. SSL encodes a casein kinase 2 (CK2) beta-subunit-like protein. Its putative ORF shares extensive (45%) homology with the genuine beta-subunit of CK2 and retains the conserved C-terminal and Glu/Asp-rich domains that are essential for CK2 holoenzyme regulation. SSL maps within region 60D1-2 of D. melanogaster and D. simulans polytene chromosomes. We present evidence that SSL was derived from the genuine betaCK2 gene by reverse transcription. This event resulted in the loss of the first three introns in the coding region of the SSL ancestor gene. Evolutionary analysis indicates that SSL has evolved under selective pressure at the translational level. Its sequence, especially in the 3' region, is much closer to the Y-linked Su(Ste) tandem repeats than to the betaCK2 gene. These results suggest that the acquisition of testis-specific autosomal genes may be important for the evolution of Drosophila as well as human Y chromosomes.

Figure 1

Diagram of Su(Ste), Stellate, and SSL gene structures and their transcripts. Structures of D. melanogaster Ste and Su(Ste) repeated genes are from refs. and , respectively. Regions of homology shared by Ste, Su(Ste), and SSL genes are shown as open rectangles. The Y-specific regions of the Su(Ste) and homologous sequence in the SSL are blackened. Hatched boxes indicate sequences of the X-specific Ste regions and homologous sequence in the SSL gene. The thick line designates the unrelated sequence in the SSL gene. The Y-specific probe used for cDNA screening is shown above the Su(Ste) unit. The black triangle indicates insertion of mobile element 1360 (out of scale). Structure of transcripts and putative sizes of gene products in kilodaltons are indicated.

Figure 2

Nucleotide and deduced amino acid sequence of the SSL gene and protein. The sequence corresponding to the longest cDNA (911) is represented by capital letters. Amino acid sequence is given in the single letter code, and the termination codon is marked with three asterisks. Differences in cDNA and genomic sequences are indicated by bold letters over (for nucleotide residues) and under (for amino acid residues) the genomic sequence. The sequence from 226 to 300 bp homologous to the 5′-untranscribed Ste region is underlined, and the sequence homologous to the Y-specific region of Su(Ste) is double underlined. The extents of other SSL cDNAs sequenced are as follows: nucleotides 356-1161, 454-1293, 454-1336 plus a 20-bp oligo(dA) tail, and 473-1301. The restriction sites (italicized) that were used for sequencing (see Materials and Methods) and the site of polyadenylylation are indicated.

Figure 3

Euchromatic location of the SSL gene. (A) In situ hybridization of the SSL-bearing cosmid 9 and of the 2.3-kb BamHI subclone to D. melanogaster and to D. simulans (Inset) polytene chromosomes, respectively. The 60D1–2 hybridization sites on chromosome 2 in both species are indicated by arrowheads. (B) Southern analysis of ≈3 μg of EcoRI-cleaved DNA from salivary glands (lane 1) and from whole flies (lane 2) probed with cDNA 911. (C) Southern analysis of 6 μg of female DNA (Batumi stock) digested with BamHI (lane 1), PstI (lane 2), and BglII (lane 3) and probed with cDNA 911. Approximate sizes of fragments are indicated in kilobases.

Figure 4

Testis-specific transcription of the SSL gene in D. melanogaster. The filters were probed with rp49 (20). (A) Total RNAs isolated from testes, embryos, larvae, pupae, adult males, and adult females (lanes 1–6, respectively) were electrophoresed in a formaldehyde gel, blotted, and hybridized with a single-stranded RNA probe complementary to the SSL plus strand. (B) The same SSL hybridization probe reveals SSL transcripts in XY and XO testes as well as the Ste transcripts of lower size in XO testes (Top, exposure time 4 days). Abundant Ste RNA was detected in XO testes using antisense Ste riboprobe (Middle, exposure time 8 h).

Figure 5

Alignment of D. melanogaster SSL, βCK2, and Stellate proteins. Identical amino acid positions shared between SSL and βCK2 or Ste are shown as shadowed boxes. The Asp/Glu-rich and C-terminal domains of the βCK2 are indicated (see text for details). Invariant residues in a cystein-rich, potential metal-binding motif CPX₃CX₂₂CPXC (29) are boxed. Positions of βCK2 introns (numbered from the beginning of the coding region of βCK2 gene) are marked with blackened triangles (introns shared by Drosophila, huma, and nematode), shadowed triangles (introns shared by Drosophila and human), or blanked triangles (Drosophila-specific introns).

Figure 6

Gene genealogy for D. melanogaster βCK2-related sequences reconstructed by the method of unweighted pair-group method (24) using the estimated numbers of synonymous substitutions. Values of synonymous divergence and approximate times of divergence are depicted (see text).