![Fig. 2. Parameters of splicing in the oocyte system. / (A) Gel analysis of a typical splicing experiment. Diagrams at sides indicate deduced structures of mRNAs from which the PCR products derive. Lane 1 shows size standards (1353, 1078, 872, 603, 310, 281, 271, 234, 194, 118, and 72 base pairs [bp], respectively). Lane 2 depicts control showing amplified β-globin pre-mRNA; lane 3, oocytes injected with β-globin pre-mRNA, showing E1/E2 spliced product; lane 4, control showing amplified 4.1 minigene pre-mRNA; lane 5, positive control showing 13/16/17 and 13/17 authentic 4.1 spliced products from differentiating MEL cells; lane 6, oocytes injected with 4.1 pre-mRNA, showing alternatively spliced 4.1 products 13/16/17 and 13/17; lane 7, oocytes mock-injected as negative control. (B) Reproducibility of the assay. Three groups of oocytes were injected with 4.1 pre-mRNA and processed in parallel. The relative efficiency of E16 inclusion among the triplicate samples, determined by densitometry and indicated above each lane, was nearly identical. (C) Time course of splicing in oocytes. Left: 50 pg of pre-mRNA was microinjected per oocyte and harvested at the indicated times for analysis. Total oocyte RNA recovery from each sample was similar (not shown). Substantial amounts of 13/16/17 and 13/17 splicing products were detected 2 hours after injection. Total spliced products remained fairly stable up to 16 hours postinjection. Right: RT/PCR products derived from endogenous frog 4.1 mRNA isoforms, representing E16 inclusion (upper band) and skipping (lower band) products as a control to demonstrate recovery of intact, amplifiable RNA from all time points. (D) Concentration dependence of splicing. Oocytes were injected with pre-mRNA as follows: Lane 1, 6 pg; lane 2, 25 pg; lane 3, 100 pg; lane 4, 400 pg; lane 5, 1.6 ng. All samples were incubated under identical conditions and harvested after 16 hours for analysis. The relative efficiency of E16 splicing given is calculated as: (inclusion products) / (inclusion plus skipping products). The efficiency varied dramatically from about 80% inclusion at low concentrations of injected substrate, to only less than 20% inclusion at high concentrations. (E) A consensus 5′ splice site mutation promotes better splicing of E16. Protein 4.1 pre-mRNA bearing the natural weak 5′ splice site yielded 55% inclusion of exon 16 (E16wt), while pre-mRNA with a strong consensus 5′ splice site exhibited 85% inclusion of exon 16 (E16↑). (Experiment performed in 2E utilized a different oocyte preparation than the remainder of this figure, thus explaining the different baseline level of wild type E16 inclusion.)](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/95/2/10.1182_blood.v95.2.692/6/bloo00218002w.jpeg?Expires=1735536975&Signature=zxWb4OIEPDCcN0LfvMEkQU5X89xg59LfmqedWELl57H0sh3NfBXOsbQhubE5XqpeiDm3i8YOScIeDVZ21eE8Bz-pKA4rYlUXvjTh4ZaI~93LNny1DzN-X4VXUpDms~IgTEY2mpNJXlOXYrX1cOMZtfrr0ndUEaXUJKl8oC65q03wKsmgy74kepNDMDi7PL24WDhbDLtFuJcNhMKwcCtGcHs1Vt7YEjsUO5HdbtM1wZC6tW3GMTJ3Ml7DzpR2wjHJmvJf4QcXpeE5zOnCcZfOtJ1QwhmrJhtEO7s5Prg9z9RASsBEkW4B~k-98BTg~DhbQ60oBPsHZEBQRdKpzrNBZQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Parameters of splicing in the oocyte system.
(A) Gel analysis of a typical splicing experiment. Diagrams at sides indicate deduced structures of mRNAs from which the PCR products derive. Lane 1 shows size standards (1353, 1078, 872, 603, 310, 281, 271, 234, 194, 118, and 72 base pairs [bp], respectively). Lane 2 depicts control showing amplified β-globin pre-mRNA; lane 3, oocytes injected with β-globin pre-mRNA, showing E1/E2 spliced product; lane 4, control showing amplified 4.1 minigene pre-mRNA; lane 5, positive control showing 13/16/17 and 13/17 authentic 4.1 spliced products from differentiating MEL cells; lane 6, oocytes injected with 4.1 pre-mRNA, showing alternatively spliced 4.1 products 13/16/17 and 13/17; lane 7, oocytes mock-injected as negative control. (B) Reproducibility of the assay. Three groups of oocytes were injected with 4.1 pre-mRNA and processed in parallel. The relative efficiency of E16 inclusion among the triplicate samples, determined by densitometry and indicated above each lane, was nearly identical. (C) Time course of splicing in oocytes. Left: 50 pg of pre-mRNA was microinjected per oocyte and harvested at the indicated times for analysis. Total oocyte RNA recovery from each sample was similar (not shown). Substantial amounts of 13/16/17 and 13/17 splicing products were detected 2 hours after injection. Total spliced products remained fairly stable up to 16 hours postinjection. Right: RT/PCR products derived from endogenous frog 4.1 mRNA isoforms, representing E16 inclusion (upper band) and skipping (lower band) products as a control to demonstrate recovery of intact, amplifiable RNA from all time points. (D) Concentration dependence of splicing. Oocytes were injected with pre-mRNA as follows: Lane 1, 6 pg; lane 2, 25 pg; lane 3, 100 pg; lane 4, 400 pg; lane 5, 1.6 ng. All samples were incubated under identical conditions and harvested after 16 hours for analysis. The relative efficiency of E16 splicing given is calculated as: (inclusion products) / (inclusion plus skipping products). The efficiency varied dramatically from about 80% inclusion at low concentrations of injected substrate, to only less than 20% inclusion at high concentrations. (E) A consensus 5′ splice site mutation promotes better splicing of E16. Protein 4.1 pre-mRNA bearing the natural weak 5′ splice site yielded 55% inclusion of exon 16 (E16wt), while pre-mRNA with a strong consensus 5′ splice site exhibited 85% inclusion of exon 16 (E16↑). (Experiment performed in 2E utilized a different oocyte preparation than the remainder of this figure, thus explaining the different baseline level of wild type E16 inclusion.)
