Na,K-ATPase in skeletal muscle: two populations of beta-spectrin control localization in the sarcolemma but not partitioning between the sarcolemma and the transverse tubules.

We used immunological approaches to study the factors controlling the distribution of the Na,K-ATPase in fast twitch skeletal muscle of the rat. Both alpha subunits of the Na,K-ATPase colocalize with beta-spectrin and ankyrin 3 in costameres, structures at the sarcolemma that lie over Z and M-lines and in longitudinal strands. In immunoprecipitates, the alpha1 and alpha2 subunits of the Na,K-ATPase as well as ankyrin 3 associate with beta-spectrin/alpha- fodrin heteromers and with a pool of beta-spectrin at the sarcolemma that does not contain alpha-fodrin. Myofibers of mutant mice lacking beta-spectrin (ja/ja) have a more uniform distribution of both the alpha1 and alpha2 subunits of the Na,K-ATPase in the sarcolemma, supporting the idea that the rectilinear sarcomeric pattern assumed by the Na,K-ATPase in wild-type muscle requires beta-spectrin. The Na,K-ATPase and beta-spectrin are distributed normally in muscle fibers of the nb/nb mouse, which lacks ankyrin 1, suggesting that this isoform of ankyrin is not necessary to link the Na,K-ATPase to the spectrin-based membrane skeleton. In immunofluorescence and subcellular fractionation experiments, the alpha2 but not the alpha1 subunit of the Na,K-ATPase is present in transverse (t-) tubules. The alpha1 subunit of the pump is not detected in increased amounts in the t-tubules of muscle from the ja/ja mouse, however. Our results suggest that the spectrin-based membrane skeleton, including ankyrin 3, concentrates both isoforms of the Na,K-ATPase in costameres, but that it does not play a significant role in restricting the entry of the alpha1 subunit into the t-tubules.

Mutations in a NIMA-related kinase gene, Nek1, cause pleiotropic effects including a progressive polycystic kidney disease in mice.

We previously have described a mouse model for polycystic kidney disease (PKD) caused by either of two mutations, kat or kat(2J), that map to the same locus on chromosome 8. The homozygous mutant animals have a latent onset, slowly progressing form of PKD with renal pathology similar to the human autosomal-dominant PKD. In addition, the mutant animals show pleiotropic effects that include facial dysmorphism, dwarfing, male sterility, anemia, and cystic choroid plexus. We previously fine-mapped the kat(2J) mutation to a genetic distance of 0.28 +/- 0.12 centimorgan between D8Mit128 and D8Mit129. To identify the underlying molecular defect in this locus, we constructed an integrated genetic and physical map of the critical region surrounding the kat(2J) mutation. Cloning and expression analysis of the transcribed sequences from this region identified Nek1, a NIMA (never in mitosis A)-related kinase as a candidate gene. Further analysis of the Nek1 gene from both kat/kat and kat(2J)/kat(2J) mutant animals identified a partial internal deletion and a single-base insertion as the molecular basis for these mutations. The complex pleiotropic phenotypes seen in the homozygous mutant animals suggest that the NEK1 protein participates in different signaling pathways to regulate diverse cellular processes. Our findings identify a previously unsuspected role for Nek1 in the kidney and open a new avenue for studying cystogenesis and identifying possible modes of therapy.

Murine recessive hereditary spherocytosis, sph/sph, is caused by a mutation in the erythroid alpha-spectrin gene.

INTRODUCTION: Spectrin, a heterodimer of alpha- and beta-subunits, is the major protein component of the red blood cell membrane skeleton. The mouse mutation, sph, causes an alpha-spectrin-deficient hereditary spherocytosis with the severe phenotype typical of recessive hereditary spherocytosis in humans. The sph mutation maps to the erythroid alpha-spectrin locus, Spna1, on Chromosome 1. MATERIALS AND METHODS: Scanning electron microscopy, osmotic gradient ektacytometry, cDNA cloning, RT-PCR, nucleic acid sequencing, and Northern blot analyses were used to characterize the wild type and sph alleles of the Spna1 locus. RESULTS: Our results confirm the spherocytic nature of sph/sph red blood cells and document a mild spherocytic transition in the +/sph heterozygotes. Sequencing of the full length coding region of the Spna1 wild type allele from the C57BL/6J strain of mice reveals a 2414 residue deduced amino acid sequence that shows the typical 106-amino-acid repeat structure previously described for other members of the spectrin protein family. Sequence analysis of RT-PCR clones from sph/sph alpha-spectrin mRNA identified a single base deletion in repeat 5 that would cause a frame shift and premature termination of the protein. This deletion was confirmed in sph/sph genomic DNA. Northern blot analyses of the distribution of Spna1 mRNA in non-erythroid tissues detects the expression of 8, 2.5 and 2.0 kb transcripts in adult heart. CONCLUSION: These results predict the heart as an additional site where alpha-spectrin mutations may produce a phenotype and raise the possibility that a novel functional class of small alpha-spectrin isoforms may exist.

An alternative first exon in the distal end of the erythroid ankyrin gene leads to production of a small isoform containing an NH2-terminal membrane anchor.

Mouse erythroid ankyrin is encoded by the Ank1 gene on Chromosome 8. The best studied isoform is 210 kDa and contains three large functional domains. We have recently reported a small Ank1 isoform (relative mobility 25 kDa) that localizes to the M and Z lines in skeletal muscle. Analyses of cDNA and genomic clones show that three transcripts of 3.5, 2.0, and 1.6 kb code for this protein. The different transcript sizes are due to their 3'-untranslated regions. They are encoded by a new first exon located in intron 39 of the Ank1 gene and three previously described Ank1 exons (40, 41, and 42). The 5'-flanking region contains a putative muscle-specific promoter. The sequence of the first 72 amino acids is novel and is predicted to form a transmembrane helix at the NH2-terminus. Functional testing of the putative transmembrane segment indicates that it acts as a membrane anchor, suggesting that the new Ank1 isoform may play an important role in organizing the contractile apparatus within the cell.

Small, membrane-bound, alternatively spliced forms of ankyrin 1 associated with the sarcoplasmic reticulum of mammalian skeletal muscle.

We have recently found that the erythroid ankyrin gene, Ank1, expresses isoforms in mouse skeletal muscle, several of which share COOH-terminal sequence with previously known Ank1 isoforms but have a novel, highly hydrophobic 72-amino acid segment at their NH2 termini. Here, through the use of domain-specific peptide antibodies, we report the presence of the small ankyrins in rat and rabbit skeletal muscle and demonstrate their selective association with the sarcoplasmic reticulum. In frozen sections of rat skeletal muscle, antibodies to the spectrin-binding domain (anti-p65) react only with a 210-kD Ank1 and label the sarcolemma and nuclei, while antibodies to the COOH terminus of the small ankyrin (anti-p6) react with peptides of 20 to 26 kD on immunoblots and decorate the myoplasm in a reticular pattern. Mice homozygous for the normoblastosis mutation (gene symbol nb) are deficient in the 210-kD ankyrin but contain normal levels of the small ankyrins in the myoplasm. In nb/nb skeletal muscle, anti-p65 label is absent from the sarcolemma, whereas anti-p6 label shows the same distribution as in control skeletal muscle. In normal skeletal muscle of the rat, anti-p6 decorates Z lines, as defined by antidesmin distribution, and is also present at M lines where it surrounds the thick myosin filaments. Immunoblots of the proteins isolated with rabbit sarcoplasmic reticulum indicate that the small ankyrins are highly enriched in this fraction. When expressed in transfected HEK 293 cells, the small ankyrins are distributed in a reticular pattern resembling the ER if the NH2-terminal hydrophobic domain is present, but they are uniformly distributed in the cytosol if this domain is absent. These results suggest that the small ankyrins are integral membrane proteins of the sarcoplasmic reticulum. We propose that, unlike the 210-kD form of Ank1, previously localized to the sarcolemma and believed to be a part of the supporting cytoskeleton, the small Ank1 isoforms may stabilize the sarcoplasmic reticulum by linking it to the contractile apparatus.

The murine mutation jaundiced is caused by replacement of an arginine with a stop codon in the mRNA encoding the ninth repeat of beta-spectrin.

The jaundiced, ja/ja, mouse mutant has a severe hemolytic anemia associated with a deficiency of beta-spectrin in erythrocyte ghosts. Genes for the disease phenotype and beta-spectrin colocalize on Chromosome 12. beta-Spectrin mRNA is not detected in reticulocytes or in brain from newborn mutant mice. To locate the nucleotide sequence alteration, the erythroid beta-spectrin transcript from mutant spleen was amplified by reverse transcription PCR and sequenced. A C-to-T alteration is present in the mutant transcript and produces a premature stop codon from an arginine codon in mRNA encoding repeat 9 of beta-spectrin at amino acid position 1160. The point mutation introduces a Dde I site that is present in PCR-amplified DNA of ja/ja and ja/+ mice but not of +/+ control mice from the strain of origin, 129/Sv, or from the two strains, WB/Re and C57BL/6J, in which the mutation has been fixed by over 53 generations of backcrossing. The genetic data confirm that the point mutation is responsible for the severe reductions in beta-spectrin mRNA of jaundiced mice.

Complete nucleotide sequence of the murine erythroid beta-spectrin cDNA and tissue-specific expression in normal and jaundiced mice.

Spectrin, a heterodimer of alpha and beta subunits, is an essential component of the red blood cell membrane skeleton. The jaundiced (ja/ja) mutation causes a severe hemolytic anemia in mice and is mapped to the erythroid beta-spectrin locus (Spnb-1) on chromosome 12. As a prerequisite for determining the molecular defect of the jaundiced mutation, we have cloned and sequenced the complete murine reticulocyte cDNA for normal Spnb-1. Two unique transcripts that differ in the placement of polyA tails are represented in the clones isolated. Amino acid sequence comparison between erythroid and murine brain spectrin (Spnb-2, chromosome 11) shows 67% identity throughout repeats 16 and 17 and complete divergence in domain III, which is associated with the alpha/beta subunit dimerization and phosphorylation. We examined the tissue distribution of normal and mutant erythroid beta-spectrin transcripts using domain-specific probes. Transcripts are detected in normal spleen tissue and reticulocytes (8 and 9.6 kb), brain tissue (10 and 11 kb), skeletal muscle tissue, and cardiac muscle tissue (11, 10.3, 7.2, and 4.0 kb). Extensive variability in mRNA processing is shown with region-specific probes. Steady state levels of the mutant transcripts are reduced when hybridized with a probe to repeats 2 through 6 with the exception of the 7.2-kb transcript that is unique to heart and skeletal muscle tissues, and is present at normal and elevated levels, respectively, in ja/ja mice. These results provide evidence for more diverse tissue-specific products of the Spnb-1 gene than were previously suspected.

Urogenital syndrome (us): a developmental mutation on chromosome 2 of the mouse.

Urogenital syndrome (us) is a recessive mutation in mice characterized primarily by abnormalities of the axial skeleton and urogenital organs. We established linkage of us with the centromeric end of Chromosome (Chr) 2, using the Robertsonian Chr Rb(2.8)2Lub. Analysis of progeny from crosses using the Chr 2 markers Danforth's short tail (Sd) and ulnaless (Ul) positioned us near two loci that have recently been mapped by RFLPs, nonerythroid alpha-spectrin (Spna-2) and the paired-box-containing-gene-8 (Pax-8). The position of us relative to these loci was established by analysis of progeny from interspecific backcrosses between the us strains and Mus spretus. The estimated map distances and most likely gene order are centromere-Pax-8-2.1 +/- 1.2-us-0.7 +/- 0.7-Spna-2; however, the reverse order cannot be ruled out. Our data make it unlikely that us is a mutation in either Spna-2 or Pax-8. Spna-2 is close enough to us, however, to be a useful marker for positional cloning of the us gene. The human mutation Nail-patella-syndrome (NPS1) maps to the region of human Chr 9 (9q34) that is homologous to the us region of mouse Chr 2. Phenotypic similarities between the two syndromes suggest the possibility that they are caused by mutations at homologous loci.

Complex patterns of sequence variation and multiple 5' and 3' ends are found among transcripts of the erythroid ankyrin gene.

The structural protein ankyrin functions in red blood cells to link the spectrin-based membrane skeleton to the plasma membrane. Ankyrin proteins are now known to occur in most cell types, and two distinct ankyrin genes have been identified (erythroid (Ank-1) and brain (Ank-2)). We have characterized transcripts of the mouse erythroid ankyrin gene by cDNA cloning and DNA sequencing. Ank-1 transcripts of 7.5 and 9.0 kilobases are found in erythroid tissues, and a 9.0-kilobase transcript is found in cerebellum. RNA hybridization blot analysis of 13 additional mouse tissues has detected four novel Ank-1 transcripts (5.0, 3.5, 2.0, and 1.6 kilobases in size). Sequencing of Ank-1 cDNA clones isolated from mouse reticulocyte, spleen, and cerebellar libraries has identified (i) multiple 5' ends that indicate possible multiple promoters; (ii) alternative polyadenylation sites that probably account for the 7.5- and 9.0-kilobase size difference; (iii) a variety of small insertions and deletions that could produce transcripts (and ultimately proteins) of nearly identical size, but different functions; and (iv) clones with large deletions of coding sequence that account for the smaller transcripts seen in spleen, skeletal muscle, and heart.

Distinct fetal Ank-1 and Ank-2 related proteins and mRNAs in normal and nb/nb mice.

Mice homozygous for the mutation normoblastosis (gene symbol nb on chromosome 8) are deficient in erythroid ankyrin (ANK-1) and have a severe hemolytic anemia throughout life. Characteristic of the disease is a dramatic decrease in the level of expression of the Ank-1 gene (chromosome 8). The other major ankyrin transcript, brain ankyrin (Ank-2 on chromosome 3) is expressed at normal levels in nb/nb mice. Surprisingly, nb/nb fetuses have normal erythrocyte counts despite the decreased levels of Ank-1 transcripts. We previously hypothesized that fetal-specific ankyrin-related proteins could exist in nb/nb fetuses to account for the lack of detrimental effects of ANK-1 deficiency. In the present report, Western and Northern blot analyses were performed on hematopoietic cells isolated from nb/nb and +/+ fetuses. An ANK-1-related protein (165 Kd) in fetal reticulocytes persisted in adult nb/nb but not in +/+ reticulocytes. An Ank-1-related transcript of 5.5 kb was found in fetal reticulocytes. This transcript appeared to be upregulated in nb/nb but not in +/+ adult reticulocytes. A fetal-specific ANK-2-related protein (155 Kd) was present in nb/nb and in +/+ fetal reticulocytes. Ank-2-related fetal liver mRNAs were present during the time the liver was actively generating erythrocytes. Neither the Ank-2-related transcripts nor the 155-Kd ANK-2-related protein were found in +/+ or mutant adult reticulocytes. The data indicate that (1) unique ankyrin-related proteins and mRNAs present in fetal erythrocytes may stabilize the ankyrin-deficient nb/nb erythrocytes and (2) adult nb/nb mice may upregulate fetal gene transcripts to compensate for the ANK-1 deficiency.

Fetal compensation of the hemolytic anemia in mice homozygous for the normoblastosis (nb) mutation.

The mouse autosomal recessive mutation nb causes a deficiency of erythroid ankyrin and generates a life-threatening hemolytic anemia in adult mice; however, at birth, nb/nb mice appear to be robust and show no pallor. In our study, the time of disease onset was sought by comparison of nb/nb and +/? mice both in utero and postnatally. Erythroid ankyrin messenger RNA (mRNA) is expressed in fetal erythroid progenitors from normal mice, but is reduced to 10% of normal levels in mutant fetuses. Despite the deficiency of erythroid ankyrin mRNA, 16 and 18 day nb/nb fetuses have normal levels of red blood cells (RBCs) and the RBCs are morphologically normal by scanning electron microscopy. The earliest signs of any clinical anomaly are an increase in the number of circulating reticulocytes and the deposition of minor amounts of iron just before birth in the 18 day fetal nb/nb liver, suggesting that RBCs are being destroyed. Within 24 hours after birth, nb/nb neonates have a slight but significant decrease of their RBC counts. During the next 5 days, the nb/nb RBC counts decrease markedly, the reticulocyte counts assume the mutant adult levels of 60%, the erythrocytes become microcytic and fragmented, and iron deposits accumulate in the liver. The rapid onset of clinical disease postnatally, coupled with our findings that the erythroid ankyrin gene is transcribed in fetal erythroid cell precursors from normal mice, suggest that mechanisms exist in the nb/nb fetus to compensate for the erythroid ankyrin deficiency.

Changing patterns in cytoskeletal mRNA expression and protein synthesis during murine erythropoiesis in vivo.

The major cytoskeletal proteins alpha-spectrin, beta-spectrin, and ankyrin are synthesized and assembled into a supportive membrane skeleton during erythroid differentiation. Information on the temporal appearance of mRNA and protein species is essential for understanding both the cytoskeletal assembly process and the function of various isoforms. We have isolated highly enriched populations of fetal erythroid cells at various stages of maturation. mRNAs for erythroid ankyrin, alpha-spectrin, and beta-spectrin were expressed at all stages but there were differences in transcript types and levels. The ratio of 9-kilobase (kb) to 7.5-kb erythroid ankyrin transcripts decreased markedly during differentiation, but there was no change in the ratio of the 10.1-kb and 9.3-kb erythroid beta-spectrin transcripts. The relative amounts of ankyrin, alpha-spectrin, and beta-spectrin mRNA increased during yolk sac cell differentiation, whereas only alpha-spectrin mRNA increased during differentiation of the fetal liver cells. The amounts of beta-spectrin mRNA exceeded the amounts of alpha-spectrin mRNA in the early precursors from both yolk sac and fetal liver; protein synthetic levels showed the same pattern. The 16-day fetal peripheral reticulocytes, on the other hand, had the adult mRNA and protein synthetic ratios with alpha/beta greater than 1. The data indicate that at least two mechanisms exist to meet changing erythroid membrane cytoskeletal requirements during development in utero: (i) stage-specific processing of the mRNA for the major cytoskeletal linker protein ankyrin and (ii) developmentally regulated alpha/beta-spectrin protein synthetic rates.

Murine erythrocyte ankyrin cDNA: highly conserved regions of the regulatory domain.

Ankyrin is an essential link between cytoskeletal proteins, such as spectrin, and membrane bound proteins, such as protein 3, the erythrocyte anion exchanger. Although the amino acid structure of human ankyrin is known, the functional regions have been only partially defined. Sequence comparisons between mouse and human ankyrin offer one mechanism of identifying highly conserved regions that probably have functional significance. We report the isolation and sequencing of a series of overlapping murine erythroid ankyrin (Ank-1) cDNAs from spleen and reticulocyte libraries (total span 6238 bp) and identify potentially important regions of murine-human reticulocyte ankyrin homology. Comparison of the predicted peptide sequences of mouse and human erythroid ankyrins shows that these ankyrins are highly conserved in both the N-terminal, protein 3 binding domain (96% amino acid identity) and in the central spectrin-binding domain (97% identity), but differ in the C-terminal regulatory domain (79% identity). However, the C-terminal regulatory domain contains two regions of peptide sequence that are perfectly conserved. We postulate these regions are important in the regulatory functions of this domain.

Purkinje cell degeneration associated with erythroid ankyrin deficiency in nb/nb mice.

Mice homozygous for the nb mutation (Chromosome 8) have a severe hemolytic anemia and develop a psychomotor disorder at 6 mo of age. The nb/nb mice are deficient in erythroid ankyrin (Ank-1) but, until the present study, the role of Ank-1 and of Ank-2 (brain ankyrin) in disease genesis was unknown. In normal erythroid tissues, we show that two major transcripts are expressed from Ank-1, and one of these is also present at high levels in the cerebellum. By in situ hybridization and immunocytochemistry, Ank-1 localizes to the cerebellar Purkinje cells and, to a lesser extent, the granule cells. In nb/nb mice, Ank-1 transcripts are markedly reduced in both erythroid and neural tissue, and nb/nb Purkinje cells and granule cells are nearly devoid of Ank-1. The neurological syndrome appears concurrently with a dramatic loss of Purkinje cells. Ank-2 maps to Chromosome 3 and its expression is unaffected by the nb mutation. We conclude that Ank-1 is specifically required for Purkinje cell stability and, in its absence, Purkinje cell loss and neurological symptoms appear.

Zinc finger protein gene complexes on mouse chromosomes 8 and 11.

Two murine homologs of the Drosophila Krüppel gene, a member of the gap class of developmental control genes that encode a protein with zinc fingers, were mapped to mouse chromosomes 8 and 11 by using somatic cell hybrids and an interspecific backcross. Surprisingly, both genes were closely linked to two previously mapped, Krüppel-related zinc finger protein genes, suggesting that they are part of gene complexes.

Ankyrin and the hemolytic anemia mutation, nb, map to mouse chromosome 8: presence of the nb allele is associated with a truncated erythrocyte ankyrin.

Mice with normoblastosis, nb/nb, have a severe hemolytic anemia. The extreme fragility and shortened lifespan of the mutant erythrocytes result from a defective membrane skeleton. Previous studies in our laboratory indicated a 50% deficiency of spectrin and an absence of normal ankyrin in erythrocyte membranes of nb/nb mice. We now report genetic mapping data that localize both the nb and erythroid ankyrin (Ank-1) loci to the centromeric end of mouse chromosome 8. Using immunological and biochemical methods, we have further characterized the nature of the ankyrin defect in mutant erythrocytes. We do not detect normal sized (210 kDa) erythroid ankyrin by immunoblot analysis in nb/nb reticulocytes. However, nb/nb reticulocytes do contain a 150-kDa ankyrin immunoreactive protein. The 150-kDa protein is present with normal-sized ankyrin in nb/+ reticulocytes but is not found in +/+ reticulocytes. Our genetic and biochemical data indicate that the nb mutation results from a defect in the erythroid ankyrin gene. A human hereditary spherocytosis putatively resulting from an ankyrin defect maps to a segment of human chromosome 8 that is homologous to the nb-ankyrin region of mouse chromosome 8. The linkage data suggest that the mouse and human diseases result from mutations in homologous loci.

Chromosomal location of three spectrin genes: relationship to the inherited hemolytic anemias of mouse and man.

Three genetic loci in the mouse affect the synthesis and assembly of the erythrocyte membrane skeleton. The spherocytosis and jaundiced loci affect the membrane skeletal protein known as spectrin. The normoblastosis locus affects the spectrin binding protein called ankyrin. We have obtained genetic data that define the linkage relationships among three spectrin genes and the spherocytosis and jaundiced loci. The erythroid alpha-spectrin gene is tightly linked to the spherocytosis locus on chromosome 1 and the jaundiced locus is on chromosome 12, tightly linked to the erythroid beta-spectrin gene. The brain alpha-spectrin (alpha-fodrin) gene is located on the centromeric end of chromosome 2 and is not closely linked to any previously mapped erythroid or neurological mutation. These results are consistent with the hypothesis that defects in the alpha- and beta-spectrin genes cause the spherocytosis and jaundiced hemolytic anemias in mice. All five loci studied are located within chromosomal segments that are conserved between mouse and man. Analysis of the data from the chromosome 12 study defines a new order for the genes on that chromosome and delineates the largest mouse/human conserved chromosomal segment yet known.

Remarkable homology among the internal repeats of erythroid and nonerythroid spectrin.

A cDNA clone for nonerythroid alpha-spectrin was identified by direct immunological screening of a chicken smooth muscle cDNA library. A library prepared in the expression plasmids pUC8 and pUC9 was screened with an antiserum specific for chicken alpha-spectrin. Blots of poly(A)+ RNA from various tissues of chicken and mouse show that the cDNA hybridizes to an 8-kilobase mRNA. The cDNA hybridizes to a single-copy sequence on Southern blots of chicken genomic DNA. The complete nucleic acid sequence of the clone has a single 1419-base open reading frame. The derived amino acid sequence is organized into two partial and three complete 106-amino-acid repeats that show homology to the repeats described for human erythroid alpha- and beta-spectrin. Immunological and biochemical data indicate that chicken nonerythroid and human erythroid alpha-spectrin are two of the more widely diverged members of the spectrin family of proteins. In this respect, the degree of homology found between them was unexpected. Our data suggest a common evolutionary origin for these two alpha-spectrins and allow some predictions concerning spectrin gene structure.

Spectrin deficient inherited hemolytic anemias in the mouse: characterization by spectrin synthesis and mRNA activity in reticulocytes.

We have investigated spectrin synthesis and mRNA activity in mice homozygous and heterozygous for six mutations occurring at three distinct loci (nb, ja, sph). When homozygous, these mutations cause severe hemolytic anemias that are characterized by specific spectrin deficiencies. Our results indicate that the primary effect of the nb mutation is a deficiency of another erythrocyte membrane skeletal protein, ankyrin. The severe deficiency of spectrin in the red blood cells of ja/ja mice is the result of a beta spectrin defect. Analysis of spectrin synthesis in mice homozygous and heterozygous for several alleles of sph indicates that the sph locus is the structural gene locus for alpha spectrin. We have mapped the sph locus to mouse Chromosome 1.