Erythroid molecular defects. (A) Northern blotting of total RNA extracted from adult reticulocytes and spleen. Note the near absence of Spna1 message in homozygous sph^3J mice when blotted with either a probe upstream or downstream of the mutation. Spna1 RNA is normal in sph^4J homozygotes. Actin and/or Gapdh expression levels were used to normalize RNA loading. (B) RT-PCR amplification of Spna1 exons 41 to 45 on total RNA extracted from adult sph^3J/+ bone marrow cells (BM) and reticulocytes (Retic.); and from E14.5 fetal liver. Amplification was followed by RsaI digestion and is shown along with undigested products. Note the production of 2 distinct products in homozygous sph^3J fetal liver. (C) Sequencing of the distinct products revealed that the C to T (bold) mutation creates a CCTTGCTGAG/gtactgggaa (over-, underlined) alternative splice (AS) site that eliminates the last 76 bp of Spna1 exon 43. (D) Sequence analysis of the H2012Y (bold) and alternative splicing predicted that AS produces 32 mutant amino acids followed by a stop codon (underlined). (E) Real time RT-PCR demonstrated that total Spnal message was 27% of NOD control in sph^3J and was only 6% of control when alternatively spliced mutant transcripts are discounted. Spna1 message levels were at control levels for heterozygous sph^3J samples. Gusb amplification was used as a housekeeping gene and was equal for all samples.