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The water flea Daphnia is a new model organism for ecological, evolutionary, and toxicological genomics. Detailed functional analysis of genes newly discovered through genomic approaches often requires overexpression of the identified protein. In the present study, we report the microinjection of in vitro-synthesized RNAs into the eggs as a method for overexpressing ubiquitous proteins in Daphnia magna. We injected a 1.3-kb mRNA that coded for the red fluorescent protein (DsRed2) flanked by UTRs from the ubiquitously expressed elongation factor 1α-1 ( EF1α- 1) into D. magna embryos. DsRed2 fluorescence in the embryos was measured 24 h after microinjection. Unexpectedly, the reporter RNA containing the 522-bp full-length EF1α- 1 3′ UTR failed to induce fluorescence. To assess reporter expression, the length of the 3′ UTR that potentially contained negative regulatory elements of protein expression, including AU-rich regions and Musashi binding elements, was serially reduced from the 3′ end. Assessing all injected RNA alternatives, mRNA containing the first 60 bp of the 3′ UTR gave rise to the highest fluorescence, 14 times the Daphnia auto-fluorescence. In contrast, mRNA lacking the entire 3′ UTR hardly induced any change in fluorescence intensity. This is the first evaluation of UTRs of mRNAs delivered into Daphnia embryos by microinjection for overexpressing proteins. The mRNA with truncated 3′ UTRs of Daphnia EF1α- 1 will be useful not only for gain-of-function analyses but also for labeling proteins and organelles with fluorescent proteins in Daphnia. [ABSTRACT FROM AUTHOR]

The epithelial Na conductance was expressed in Xenopus laevis oocytes by injection of size-fractionated mRNA of bovine tracheal epithelium. Fractionation was achieved by sucrose density gradient centrifugation. Successful expression was analysed by recording current/voltage ( I/V) curves in the presence and absence of amiloride (10 μmol/l). The newly expressed conductance was half-maximally inhibited by 44 nmol/l amiloride and exhibited a selectivity for Na over K of 140∶1. I/V curves obtained at different extracellular Na concentrations ([Na]) were subjected to a Goldman-fit analysis to obtain the relation between Na permeability ( P) and [Na]. The data show that decreasing [Na] from 85 mmol/l to 0.85 mmol/l increased P by more than threefold, which is thought to reflect Na channel inhibition by increasing [Na]. This effect clearly exceeded what can be attributed to concentration saturation of single Na channel conductance (Palmer and Frindt (1986) Proc Natl Acad Sci USA 83:2767). No correlation of inhibition with intracellular Na concentration was observed. Preservation of the [Na]-dependent self-inhibition by the newly expressed Na conductance suggests that it is an intrinsic property of the Na channel protein, probably mediated by an extracellular Na binding site. [ABSTRACT FROM AUTHOR]