Evolution of hydra, a recently evolved testis-expressed gene with nine alternative first exons in Drosophila melanogaster

"We describe here the Drosophila gene hydra that appears to have originated de novo in the melanogaster subgroup and subsequently evolved in both structure and expression level in Drosophila melanogaster and its sibling species. D. melanogasterhydra encodes a predicted protein of ~300 amino acids with no apparent similarity to any previously known proteins. The syntenic region flanking hydra on both sides is found in both D. ananassae and D. pseudoobscura, but hydra is found only in melanogaster subgroup species, suggesting that it originated less than ~13 million years ago. Exon 1 of hydra has undergone recurrent duplications, leading to the formation of nine tandem alternative exon 1s in D. melanogaster. Seven of these alternative exons are flanked on their 3' side by the transposon DINE-1 (Drosophila Interspersed Element-1). We demonstrate that at least four of the nine duplicated exon 1s can function as alternative transcription start sites. The entire hydra locus has also duplicated in D. simulans and D. sechellia. D. melanogasterhydra is expressed most intensely in the proximal testis, suggesting a role in late-stage spermatogenesis. The coding region of hydra has a relatively high Ka/Ks ratio between species but the ratio is less than one in all comparisons, suggesting that hydra is subject to functional constraint. Analysis of sequence polymorphism and divergence of hydra shows that it has evolved under positive selection in the lineage leading to D. melanogaster. The dramatic structural changes surrounding the first exons do not affect the tissue specificity of gene expression: hydra is expressed predominantly in the testes in D. melanogaster, D. simulans and D. yakuba. However, we have found that expression level changed dramatically (~>20 fold) between D. melanogaster and D. simulans. While hydra initially evolved in the absence of nearby transposable element insertions, we suggest that the subsequent accumulation of repetitive sequences in the hydra region may have contributed to structural and expression-level evolution by inducing rearrangements and causing local heterochromatinization. Our analysis further shows that recurrent evolution of both gene structure and expression level may be characteristics of newly evolved genes. We also suggest that late-stage spermatogenesis is the functional target for newly evolved and rapidly evolving male-specific genes."

use transition/transversion (κ) ratio test to detect functional polypeptide

"To confirm our results using an independent nucleotide-based approach (as opposed to the codon-based test described earlier), we applied the transition/transversion (κ) ratio test to make inferences about biological significance of ARFs. The test is based on the following reasoning: in most standard protein-coding regions (with only one reading frame), κ at the third codon position (κ3) is significantly different (higher) than at the first and second codon positions (κ12), so that κ12 < κ3 [15]. This is because most substitutions at the third codon position are synonymous, whereas in the first codon position all but eight substitutions are nonsynonymous, and all substitutions in the second codon position are nonsynonymous. By contrast, in overlapping reading frames, codon positions are codependent. For example, in a +1 ARF, the third codon positions correspond to the first codon positions of the canonical frame. Thus, almost every change in the third codon position of the ARF is guaranteed to change amino acids encoded in the canonical frame. However, if the ARF encodes a truly functional product, purifying selection would resist such changes, and the condition κ12 < κ3 would not hold. This gives us the opportunity to test functionality of ARF in our dataset by contrasting two hypotheses: H₀: κ₁₂ = κ₃ (ARF does encode functional polypeptide) and H_A: κ₁₂ < κ₃ (ARF does not encode functional polypeptide). To perform this test, we used a maximum likelihood framework to test κ₁₂ and κ₃ for equality [16]. Application of the test to our list of dual-coding genes identified 18 candidates" PLoS Computational Biology - A First Look at ARFome: Dual-Coding Genes in Mammalian Genomes