4112 Nucleic Acids Research, 2011, Vol. 39, No. 10 After several washes for 1 h in 0.1 SSC, 10 mmol/l dithiothreitol at 65 C, slides were dehydrated through an ethanol series, dipped in NTB2 emulsion (Kodak; diluted 1:1 with H2O) and exposed at 4 C for 3–7 days. After development, nuclear spreads were stained with Coomassie blue R. The cDNA used as a template for the synthesis of RBMX probes was isolated from the Xenopus tropicalis (xthr) library of cDNAs prepared from the head and retinas of advanced limb bud stages (28). The xthr17M05 plasmid contained a partial cDNA of RBMX (830 bp) inserted into the pCMVSPORT6-xthr plasmid, a modiﬁed version of the PCMVsport6 vector. A small portion of this RBMX cDNA (300 bp) was subcloned into the pCR-Blunt II-TOPO vector (Invitrogen). Both of the long and the small cDNAs of RBMX were used for the synthesis of the RNA probes. The plasmids were linearized with EcoR1 or BamH1. The sense and antisense RBMX riboprobes (SA. 3 108 cpm/ mg) were generated by in vitro transcription using the Riboprobe in vitro Transcription system from Promega and [35S] UTP. RESULTS Evidence for a 42 kDa polypeptide(s) with sequence-speciﬁc RNA-binding activity linked to the sex genotype The WEc RNA probe was used in NWA to identify nuclear proteins mediating its binding to the W chromo- some. Proteins extracts of GVs of ZZ, ZW and WW oocytes were separated by mono-dimensional electrophor- esis in polyacrylamide gels and transferred to nitrocellu- lose membranes. Membranes were probed with 32P-labelled RNA probes. In parallel to the WEc RNA, other RNA probes, which did not label the W chromo- some lampbrush loops, were used as controls (Figure 1). In repeated NWA experiments, the WEc RNA probe showed a very similar pattern of binding proteins for the three karyotypes except in the 42 kDa range where strong binding signals were present for the ZW and WW GVs while no signal was observed for the ZZ GVs. The PB6 RNA probe showed the same differential interaction with the 42 kDa polypeptide(s) for the ZW and WW GVs although the binding signals were weaker than the ones Figure 1. Pleurodeles waltl 42 kDa polypeptide(s) bind the WEc RNA speciﬁcally in the ZW and WW GVs. (A) Restriction map of the clone 130 from which the RNA probes were derived by in vitro transcription. (B) NWA of 32P-labelled RNA probes with nuclear proteins from ZZ, ZW and WW GVs. Nuclear proteins were transferred to nitrocellulose membrane after electrophoresis in mono-dimensional polyacrylamide gels and incubated with the RNA-probes. Note that nuclear protein(s) in the 42 kDa range speciﬁcally bound to the WEc and PB6 RNA probes in ZW and WW karyotypes and not in ZZ karyotypes (arrow and box). No interaction could be detected for these proteins with the other RNA probes tested.