Figure 5.

Amplification of regions between introns 4 and 6 in unspliced or partially spliced COL5A1 precursor mRNA (cDNA) isolated from cultured fibroblasts after incubation with actinomycin D (Act.D) for 0, 5, 10, 20, 40, and 60 min. A, Amplification with an intron 4 (IN 4S) and exon 6 (E 6A) primer pair. The full-length product was quickly lost in the control cells but remained present for 60 min in the patient cells. In both cell strains, the shorter product, from which intron 5 had been removed, persisted. B and C, PstI restriction fragments of excised and reamplified full-length (intron 4–exon 5–intron 5–exon 6) and partially spliced (intron 4–exon 5–exon 6) products, respectively, derived from the PstI^+/− control individual (C) and the PstI^+/− patient (P). In the patient cells, the full-length product (B) contained mRNA derived only from the mutant (PstI⁻) allele, and the product from which intron 5 had been removed (C) was derived largely from the mutant allele. D, With an intron 4 (IN 4S mod) and intron 6 (IN 6A) primer pair, the amount of full-length product rapidly declined in the control cells but remained abundant in the patient cells. The partially spliced product, which removed intron 5 but contained introns 4 and 6, remained. E, PstI restriction fragments of excised and reamplified partially spliced (intron 4–exon 5–exon 6–intron 6) product derived from the PstI^+/− control individual (C) and patient (P). In the patient cells, this product was derived from the mutant and normal alleles in approximately equal amounts. Molecular weight marker is λ-DNA cut with PstI. gen = genomic DNA control individual; NC = PCR-reagent control individual; +/+, +/−, and −/− = PstI-restricted cDNAs reverse transcribed from control RNA samples from individuals who were homozygous positive, heterozygous, and homozygous negative, respectively, for a polymorphic PstI site in COL5A1 exon 5.