Preparation of a highly active cell-free translation system from immature Xenopus laevis oocytes

Abstract

Understanding mechanisms of post-transcriptional control of gene expression has come under much scrutiny in recent years. A key question in this field is how the translation of specific mRNAs is activated or repressed both spatially and temporally in a given cell. In oocytes of the frog Xenopus laevis a number of mRNAs are localized early in oogenesis and subsequently translated at later stages. We have developed a highly active cell-free translation system from oocytes in the early stages of oogenesis that is applicable to the study of translation and translational control of both endogenous and exogenous mRNAs.

Figure 1

Procedure for preparation of a translationally active oocyte lysate (XTL). Stage I–III oocytes are separated from stage IV–VI oocytes by passage through a 500 μm sieve, followed by manual removal of any pigmented oocytes that pass through the sieve (black arrow heads). Stage I–III oocytes are washed into a hypotonic lysis buffer (HLB), disrupted by dounce homogenization, and centrifuged to remove cellular debris. The supernatant (XTL) is removed and used for in vitro translation.

Figure 2

Activity of the XTL translation lysate. (A) Time course of incorporation of [³⁵S]-methionine into protein using XTL prepared as described in section 2.1.2. Translation assays were performed as described in section 2.1.2. At each time point, incorporation of the radiolabel was measured by TCA precipitation of a 10 μl reaction and subsequent binding to a 24 mm GF/A Whatman glass microfiber filter. [³⁵S]-methionine incorporation (cpm, y-axis) is plotted against time (minutes, x-axis). (B) Translation efficiency of the Xenopus translation lysate (XTL) was compared to that of Rabbit reticulocyte lysate (RRL). XTL and RRL were programmed with RNA transcribed from pSP64-XLucXβ (Fig. 3A), and incubation of XTL without RNA (no RNA) served as a negative control. Analysis of luciferase activity demonstrates that XTL translation activity is greater than that of the commercially available (Promega) RRL.

Figure 3

Schematic of pSP64-Luc reporters for testing UTR-mediated translation regulation. (A) pSP64-XβLucXβ contains the firefly luciferase gene (white arrow) flanked by Xenopus β-globin 5′ (black) and 3′ (gray) untranslated region (UTR) sequences. The positions of key restriction sites are marked. (B) 5′ UTR sequences of interest can be inserted into the HindIII site (marked by a white arrowhead) of pSP64-LucXβ. (C) 3′ UTR sequences of interest can be inserted into the XbaI site (marked by a white arrowhead) of pSP64-XβLuc. For the production of run-off transcripts all plasmids (A–C) are linearized with EcoRI and transcribed using SP6 RNA polymerase; the SP6 promoter regions are marked at the left.