Nucleic Acids Research, 2011, Vol. 39, No. 7 2711 Figure 8. (A) RT-PCR analysis of rpL22-like transcripts using primers that bridge exons in rpL22-like and rpL22-like short. RT-PCR products using the exon1/2 bridge primer should hybridize to rpL22-like and rpL22-like short, producing bands of 950 bp (arrow) and 392 bp (closed arrowhead), respectively (red primer set in Figure 7). All samples show such products in multiple experiments. Other amplicons (<350 bp) were cloned and sequenced and determined to be the result of non-speciﬁc ampliﬁcation. The novel rpL22-like short exon2/3 bridge primer should speciﬁcally hybridize only to rpL22-like short (purple primer set in Figure 7). The expected amplicon of 370 bp (open arrowhead) is seen in all samples, conﬁrming the presence of rpL22-like short. E: embryo; L: larval; A: adult; S2: S2 cells; M: pGEM marker. (B) Northern blot analysis of rpL22-like mRNAs from different developmental stages. PolyA+ RNA from embryonic, larval and adult ﬂy stages was probed with 32P-labelled rpL22-like- speciﬁc cDNA oligomers. Estimated transcript sizes based on RNA markers are shown in kilobases (kb). rpL22-like mRNA is predicted to be 1.194 kb (Flybase). rpL22-like short mRNA is predicted to be a minimum size of 0.625 kb. Arrows highlight prominent transcripts detected in all stages. (C) Embryonic PolyA+RNA was probed with 32P-labelled rpL22-like-speciﬁc cDNA oligomers [(full speciﬁc—lane1) or (ﬂanking—lane 2)]. RNA sizes were determined relative to an RNA marker. bands represent degradation products derived from RpL22-like is unclear; however, a computational investi- gation of proteolytic sites that would be found in the Drosophila RpL22-like amino acid sequence (FlyBase ID: FBgn0034837) does not predict degradation products of either MW (PeptideCutter, Expasy.org). A deﬁnitive resolution of this issue was not addressed in this study, but will be addressed by MALDI-TOF analysis (in progress). The relatively low abundance of the 13 kDa protein (estimated based on exposure times required to visualize RpL22-like short compared to RpL22-like; Figure 9B and C) is similar to what might be expected if RpL22-like short protein expression (when compared to RpL22-like expression) is approxi- mately proportional to the amounts of rpL22-like short and rpL22-like mRNAs in testis. DISCUSSION RpL22-like is a tissue-speciﬁc RP As a strategy for biochemical characterization of RpL22 from tissues in which the protein is abundant, we reﬁned the expression pattern for rpL22-like in several tissues and developmental stages, showing that both rpL22-like mRNA and its protein product are highly enriched in adult testes compared to other tissues as analysed by qRT-PCR and western analysis, respectively. Within the male reproductive tract, RpL22-like expression is limited to testes, as no other reproductive organs showed expres- sion, as visualized by IHC. In a microarray study evaluating testis-speciﬁc paralogue gene expression in general, Mikhaylova et al. (27) identiﬁed RpL22 as one of 12 down-regulated RP