Tissue Expression and Actin Binding of a Novel N-Terminal Utrophin Isoform
Figure 2
Detection of the mRNA for the N-utrophin isoform and for full-length Up395 in rat tissues by RT-PCR and electrophoresis in 1% agarose with ethidium bromide. (a) Organisation of mRNA for full-length utrophin (upper line) and for N-utrophin (lower line) with direction of primers (lettered arrows) and exon borders (dashed lines). Sequence homolgy breaks down at nucleotide (nt) 1803. Non-discriminating primers (A, B, C, D) recognise both isoforms, while primers (E, F, G) are specific for N-utrophin, and primers (H, J) are specific for full-length utrophin. The genomic DNA contains intron-13 of 1.2 kilobases (kb) between exon-13 and 14. Exon numbering is in homology to that of dystrophin. (b) RT-PCR (40 cycles) products ranging in size from 100 to 500 base pairs (bp) from poly(A)+ RNA of rat kidney primed with either oligo(dT) or pd(N)6. (c) RT-PCR (40 cycles) products (AF and AG) from genomic rat liver DNA display sizes of 1.5 and 1.7 kb containing intron-13 which adds 1.2 kb to each fragment. Product (EG) of 0.28 kb is specific for N-utrophin as control. The three black lanes between EG and the markers (M) are controls with the same primer pairs but without DNA. (d) RT-PCR (30 cycles) products (EG specific for N-utrophin and HJ specific for full-length utrophin) in different tissues. Black lanes are negative controls without reverse transcriptase. Note the larger amount of N-utrophin message in skeletal muscle of young rats (postnatal day 8) as compared to adult (arrow). Pairs of letters denote primer pairs; M stands for DNA markers.
