Nucleic Acids Research, 2011, Vol. 39, No. 7 2705 Figure 1. (A) Clustal W alignment of D. melanogaster RpL22 and RpL22-like sequences. Aligned sequences (RpL22: FBgn0015288; RpL22-like: FBgn0034837) show conservation at the C-terminus (46% sequence identity; 73% sequence similarity), but divergence at the N-terminus (sequence identity 36%; sequence similarity 47%). Overall amino acid identity is 37% (6). Shaded amino acid residues mark conservation within the RpL22e superfamily. Other residues deﬁne the N-terminal extension with homology to histone H1 [(7) for RpL22 and RpL23a]. Boxes highlight functional residues involved in nuclear localization (box 1), rRNA binding (boxes 2 and 3) and nucleolar localization (boxes 2 and 3) (20). Underlined residues in the C-termini were used to make polyclonal peptide Abs. (B) Translation of rpL22-like short mRNA (GenBank accession no. HM756190) using Translation Tool (expasy.org) revealed a putative protein of 123 amino acid, consisting primarily of the histone H1-like domain (N-terminus) and not the RpL22-like domain (C-terminus). Underlined residues in the C-termini were used to make polyclonal peptide Abs. expression analyses (FlyAtlas: ﬂyatlas.org), showing that rpL22-like mRNA is highly enriched ( 4300-fold in this study) in testis compared with ovary (Supplementary Table S2). Using the testis-speciﬁc 2-tubulin gene (22) and the germline-speciﬁc Vasa gene [males: (18); females: (23)] for comparative purposes in the ﬂy gonad, mRNA levels seen for rpL22-like short (and for rpL22-like) in tissues other than in testis may be due to basal level tran- scription (Figure 3) as 2-tubulin mRNA [widely regarded as a testis-speciﬁc protein, e.g. (22,24)] has some level of detection even in ovaries. While found at 9800-fold lower levels compared with rpL22-like in testes, all samples showed that the shorter isoform is not the prominent isoform, with highest and