Identification of sph^3J and sph^4J defects in Spna1. (A) A cytosine to thymine change in the sph^3J allele causes a histidine to tyrosine substitution at residue 2012 of α-spectrin, and a guanine to adenine change in the sph^4J allele causes a cysteine to tyrosine substitution at residue 2384 of α-spectrin. (B) Schematic representation of the α-spectrin monomer is depicted with repeat 10 shown as a Src homology domain (SH3; redrawn from Walensky et al⁶ ). The approximate locations of 4 previously identified sph alleles (sph, sph^Dem, sph^1J, sph^2BC), sph^3J and sph^4J, and the sph^Ihj allele are indicated. sph has a single nucleotide deletion in repeat 5 that results in a premature stop codon⁴³ ; sph^Dem has an in-frame deletion of exon 11 that deletes 46 amino acids from repeat 5²¹ ; sph^2BC has a guanine to thymine substitution in intron 41 that results in a frame shift and premature stop codon in repeat 19⁴² ; sph^1J is a deletion of the C-terminal 13 amino acids⁴² ; sph^3J is a missense mutation in repeat 19 of the αβ-spectrin dimer nucleation site; sph^4J is a missense mutation in the C-terminus within the EF-hand domain just upstream of sph^1J deletion; and sph^Ihj is a premature stop codon in repeat 18. (C) The sph^3J mutation creates an RsaI recognition site that was used to genotype mice after PCR amplification. The wild-type fragment is 536 bp. RsaI digestion produces fragments of 401 and 135 bp in mutant mice. (D) The sph^4J mutation destroys an RsaI site and was used to genotype mice after PCR amplification. RsaI digestion does not produce the wild-type fragments of 315 and 105 bp but instead a 420-bp fragment. Note that the smallest PCR fragments, 135 bp and 105 bp, are not visible. All inbred strains of mice tested for each allele retain the wild-type genotypes.