Figure 2. Expression of synthetic mRNAs af. ter oocyte injection. {A) Schematic representa· tion of wild-type and mutated BMP and ac· tivin molecules. The RXXR amino acid cleav· age site sequences in the BMP and acti vin molecules were mutated to GVDG in the cleavage mutant constructs. Native molecules form dimers {the disulfide bond is indicated), which are then cleaved to form the carboxyterminal mature ligand {shaded region) and the amino-terminal precursor remainder pep· tide (hatched region). The cleavage mutants do not mature; thus, oocytes injected with the cleavage mutant constructs only generate the high molecular weight uncleaved polypeptide. The relative positions of HA and Flag epitopes within the pro-domain are indicated. (B) In vivo translation of wild-type and cleavage mutant mRNAs. Media from oocytes injected with 50-200 ng of the indicated mRNAs were pulse labeled (3 hrs) with (35S)methionine and then analyzed by 14% SDS-PAGE. under reducing conditions. Wild-type activin is cleaved to form two polypeptides (37 and 14 kD) {asterisks, lane 21, whereas Cm- activin is not {51 kD) (asterisk, lane 3). Both XBMP-7 and XBMP-4 mRNA·injected oocytes produced cleaved amino-terminal peptides [34 and 35 kD, respectively) {asterisks, lanes 4,6). HA- Cm- XBMP-7 and Cm-XBMP-4 mRNA· injected oocytes failed to generate the cleaved polypeptides and produced only the uncleaved form (S 1 and 48 kD, respectively) (asterisks, lanes 5,7). Medium from HA- Cm- XBMP-7-injected oocytes was immunoprecipitated before SDS·PAGE {lane 5). Uninjected oocytes are shown in lane 1. HA·tagged Cm- XBMP-7 was found to have activity similar to Cm- XBMP-7. (C) Maturation of FlagXBMP · 7 and Flag- XBMP-4 proteins is inhibited by expression of Cm-XBMP-7. (Lane 1) Uninjected oocytes; (lane 2) HA- Cm-XBMP-7 mRNA {50 ng)·injected oocytes. {Lanes 3 and 4, respectively) Injection of Flag- XBMP-7 or Flag- XBMP-4 mRNA {SOng) results in the formation of cleaved precursor remainder peptides (asterisk at 34 and 3S kD, respectively) in addition to uncleaved monomers (arrowhead at 51 and 48 kD, respectively) and uncleaved dimers farrow at 102 and 96 kD, respectively). {Lanes 5 and 6, respectively) Coinjection of Cm- XBMP-7 mRNA (200 ng) with Flag- XBMP-7 mRNA ISO ng) or Flag-XBMP-4 mRNA ISO ng) results in a decreased amount of precursor remainder peptide. This indicates that processing of Flag-XBMP· 7 and Flag-XBMP-4 is inhibited in the presence of Cm- XBMP-7. 1Lanes 7 and 8, respectively) Coinjection of Cm- activin mRNA (200 ng) with Flag-XBMP-7 mRNA (SOng) or Flag- XBMP-4 mRNA ISO ng) does not significantly reduce the formation of the precursor remainder peptide, indicating that the inhibition of processing is specific to the BMPs. Flag-tagged XBMP-7 and XBMP-4 were found to have similar, although slightly weaker, activity compared to native XBMP· 7 and XBMP-4. All samples are run under nonreducing conditions. All lanes are immunoprecipitated with either the HA flane 2 only) or Flag antibodies.
Image published in: Hawley SH et al. (1995)
Copyright © 1995. Image reproduced on with permission of the Publisher, Cold Spring Harbor Laboratory Press. This is an Open Access article.
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