Fig. 1.
Fig. 1. Analysis of the translational efficiency of normal and mutant TPO transcripts in reticulocyte lysates. (A) Exon composition and ORFs of the normal and mutant TPO mRNAs originating from promoter 2 (P2). Exons are drawn as numbered boxes and the TPO protein coding region is shaded. The position of the single G nucleotide deletion is indicated (▵G). The patterns of uORFs are drawn separately for normal and mutant TPO mRNA. The uAUG codons (•) are placed in the 3 possible reading frames (Roman numbers) and numbered in the order in which they appear in the full-length P1 transcript. The resulting uORFs are shown as horizontal lines and the position of stop codons is indicated by short vertical lines. Thick solid lines with arrowheads represent the normal and ▵G-mutant TPO-ORFs, with the corresponding amino acid sequences indicated below. Initiator methionines are highlighted by black boxes. (B) In vitro transcription-translation analysis of mRNAs originating from P2. Equal amounts of in vitro-transcribed TPO mRNA variants (lower panel) were translated in vitro in reticulocyte lysate in the presence of 35S-methionine (upper panel). ▵UTR, mRNA with deletion of the entire 5′-UTR; n, normal mRNA; ▵G, ▵G-mutant mRNA. The protein bands in the upper panel were AUG8, the normal TPO protein initiated at the physiological start site; AUG7, mutant TPO protein originating from AUG7; asterisk, cryptic non-AUG initiation in exon 3. (C) TPO secretion by COS cells transfected with either the normal (n) or ▵G-mutant (▵G) TPO cDNA. TPO protein concentration in COS cell supernatants was determined by ELISA (▪) and by bioassay (□). Error bars represent the standard deviations. co, supernatant from nontransfected COS cells. The abundance of transfected TPO mRNA was assessed by Northern blot. To confirm equal transfection efficiencies, mRNA for vector-derived neomycin resistance gene (neo) was determined by reprobing of the Northern blot and equal RNA loading was verified by visualizing the 18S ribosomal RNA with ethidium bromide staining (lower panels).

Analysis of the translational efficiency of normal and mutant TPO transcripts in reticulocyte lysates. (A) Exon composition and ORFs of the normal and mutant TPO mRNAs originating from promoter 2 (P2). Exons are drawn as numbered boxes and the TPO protein coding region is shaded. The position of the single G nucleotide deletion is indicated (▵G). The patterns of uORFs are drawn separately for normal and mutant TPO mRNA. The uAUG codons (•) are placed in the 3 possible reading frames (Roman numbers) and numbered in the order in which they appear in the full-length P1 transcript. The resulting uORFs are shown as horizontal lines and the position of stop codons is indicated by short vertical lines. Thick solid lines with arrowheads represent the normal and ▵G-mutant TPO-ORFs, with the corresponding amino acid sequences indicated below. Initiator methionines are highlighted by black boxes. (B) In vitro transcription-translation analysis of mRNAs originating from P2. Equal amounts of in vitro-transcribed TPO mRNA variants (lower panel) were translated in vitro in reticulocyte lysate in the presence of 35S-methionine (upper panel). ▵UTR, mRNA with deletion of the entire 5′-UTR; n, normal mRNA; ▵G, ▵G-mutant mRNA. The protein bands in the upper panel were AUG8, the normal TPO protein initiated at the physiological start site; AUG7, mutant TPO protein originating from AUG7; asterisk, cryptic non-AUG initiation in exon 3. (C) TPO secretion by COS cells transfected with either the normal (n) or ▵G-mutant (▵G) TPO cDNA. TPO protein concentration in COS cell supernatants was determined by ELISA (▪) and by bioassay (□). Error bars represent the standard deviations. co, supernatant from nontransfected COS cells. The abundance of transfected TPO mRNA was assessed by Northern blot. To confirm equal transfection efficiencies, mRNA for vector-derived neomycin resistance gene (neo) was determined by reprobing of the Northern blot and equal RNA loading was verified by visualizing the 18S ribosomal RNA with ethidium bromide staining (lower panels).

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