Fig. 4.

Translation of nanos1 RNA is blocked at initiation. (A) Translation of wild-type (WT) nanos1 and insertion mutants in Xenopus oocytes. WT nanos1 and TCEΔare described in Fig. 2B and the insertion mutants are as illustrated. Nucleotides from equivalent positions of β-globin were inserted in between the start codon and the TCE. Translation products from injected oocytes were analyzed by blotting with anti-Nanos1 antibody. (B) Translation of nanos1 and nanos1 mutants in wheat germ extracts. One microgram of synthetic mRNA was translated in wheat germ extracts and the resulting products analyzed as in A. (C-E) Ribosomal loading assay performed with radiolabeled Myc-nanos1, nanos1, nanos1-TCEΔ or nanos1-15nt-insertion (nanos1-I15) transcripts. Transcripts were incubated in wheat germ extract in the presence of cycloheximide (500 μg/ml) (C,D) or GMP-PNP (2 mM) (E) at 18°C for 15 minutes and the reaction mixture fractionated on 10-30% linear sucrose gradients. The labeled mRNA in the fractions is expressed as a percentage of total counts recovered and is plotted against the fraction number. The dashed line denotes the A₂₅₄ absorption profile. Compare the regions indicated by the white arrows and black arrowheads (see text).