Cloning and analysis of cDNA clones for rat kidney alpha-spectrin

We have isolated a 3922-base pair (bp) cDNA clone for rat nonerythroid alpha-spectrin from a rat kidney lambda gt11 cDNA library. Sequence analysis revealed that this cDNA contains an open reading frame of 3090 bp encoding for the C-terminal 1030 amino acid sequence of rat kidney alpha-spectrin. The 3'-untranslated region (including a 38-bp poly(A+) tail) contains an 832-bp sequence. A single mRNA of about 8 kilobase pairs was detected in rat liver, kidney, brain, heart, intestine, lung, testis, stomach, spleen, and muscle with varying abundances, which is consistent with and further confirms the presence of spectrins in nonerythroid tissues as demonstrated previously by immunoblot analysis. Southern blot analysis suggested that there is a single gene for nonerythroid alpha-spectrin. The derived amino acid sequence contains sequence from the spectrin 106-residue internal repeat 12 to the C terminus of rat kidney alpha-spectrin. Sequence comparison with human and chicken nonerythroid alpha-spectrin showed that nonerythroid alpha-spectrin is well conserved during evolution. The rat kidney alpha-spectrin sequence, when compared to rat brain alpha-spectrin, contains an extra 76-amino-acid sequence at the C terminus. Sequence comparison of all the internal repeats available revealed that the internal repeat 3, 4, 5, 6, 7, and 8 has highest sequence similarity with internal repeat 12, 13, 14, 15, 16, and 17, respectively. Therefore, internal repeats 3-8 and 12-17 are most likely derived from an ancestral gene through gene duplication, suggesting that the spectrin gene is derived from a half-spectrin gene by gene duplication and divergence during evolution.     

Comparison of nonerythroid alpha-spectrin genes reveals strict homology among diverse species.

The spectrins are a family of widely distributed filamentous proteins. In association with actin, spectrins form a supporting and organizing scaffold for cell membranes. Using antibodies specific for human brain alpha-spectrin (alpha-fodrin), we have cloned a rat brain alpha-spectrin cDNA from an expression library. Several closely related human clones were also isolated by hybridization. Comparison of sequences of these and other overlapping nonerythroid and erythroid alpha-spectrin genes demonstrated that the nonerythroid genes are strictly conserved across species, while the mammalian erythroid genes have diverged rapidly. Peptide sequences deduced from these cDNAs revealed that the nonerythroid alpha-spectrin chain, like the erythroid spectrin, is composed of multiple 106-amino-acid repeating units, with the characteristic invariant tryptophan as well as other charged and hydrophobic residues in conserved locations. However, the carboxy-terminal sequence varies markedly from this internal repeat pattern and may represent a specialized functional site. The nonerythroid alpha-spectrin gene was mapped to human chromosome 9, in contrast to the erythroid alpha-spectrin gene, which has previously been assigned to a locus on chromosome 1.     

Erythroid expression of the human alpha-spectrin gene promoter is mediated by GATA-1- and NF-E2-binding proteins

alpha-Spectrin is a highly expressed membrane protein critical for the flexibility and stability of the erythrocyte. Qualitative and quantitative defects of alpha-spectrin are present in the erythrocytes of many patients with abnormalities of red blood cell shape including hereditary spherocytosis and elliptocytosis. We wished to determine the regulatory elements that determine the erythroid-specific expression of the alpha-spectrin gene. We mapped the 5' end of the alpha-spectrin erythroid cDNA and cloned the 5' flanking genomic DNA containing the putative alpha-spectrin gene promoter. Using transfection of promoter/reporter plasmids in human tissue culture cell lines, in vitro DNase I footprinting analyses, and gel mobility shift assays, an alpha-spectrin gene erythroid promoter with binding sites for GATA-1- and NF-E2-related proteins was identified. Both binding sites were required for full promoter activity. In transgenic mice, a reporter gene directed by the alpha-spectrin promoter was expressed in yolk sac, fetal liver, and erythroid cells of bone marrow but not adult reticulocytes. No expression of the reporter gene was detected in nonerythroid tissues. We conclude that this alpha-spectrin gene promoter contains the sequences necessary for low level expression in erythroid progenitor cells.     

Generation of diversity in nonerythroid spectrins. Multiple polypeptides are predicted by sequence analysis of cDNAs encompassing the coding region of human nonerythroid alpha-spectrin

Nonerythroid alpha-spectrin (alpha-fodrin) is a major component of the membrane skeleton in diverse cell types. Overlapping cDNAs have been isolated which encompass the coding region of human lung fibroblast nonerythroid alpha-spectrin. The composite sequence of 7,787 nucleotides encodes a polypeptide of 2,472 amino acids (predicted Mr of 283,964). This sequence has 58% amino acid identity with human erythroid alpha-spectrin, which is encoded on a different gene, and 96% amino acid identity with the full-length sequence of chicken brain alpha-spectrin. We previously reported the variable expression in human fibroblast alpha-spectrin of 20 amino acids between repeats 10 and 11 (McMahon, A. P., Giebelhaus, D. H., Champion, J. E., Bailes, J. A., Lacey, S., Carritt, B., Henchman, S. K., and Moon, R. T. (1987) Differentiation 34, 68-78). In this study, we report additional heterogeneity in fibroblast alpha-spectrin near the carboxyl-terminal end. One of the fibroblast cDNAs (clone 3D) has an in-frame deletion of 18 nucleotides within spectrin repeat 21 when compared to an overlapping fibroblast cDNA (clone 7). As this heterogeneity in amino acid sequence occurs near domains of nonerythroid alpha-spectrin suggested to bind calcium or actin, it is possible that fibroblasts express functionally distinct isoforms of nonerythroid alpha-spectrin.     

Drosophila spectrin. II. Conserved features of the alpha-subunit are revealed by analysis of cDNA clones and fusion proteins

Drosophila alpha-spectrin cDNA sequences were isolated from a lambda gt11 expression library. These cDNA clones encode fusion proteins that include portions of the Drosophila alpha-spectrin polypeptide as shown by a number of structural and functional criteria. The fusion proteins elicited antibodies that reacted strongly with Drosophila and vertebrate alpha-spectrins and a comparison of cyanogen bromide peptide maps demonstrated a clear structural correspondence between one fusion protein and purified Drosophila alpha-spectrin. Alpha-spectrin fusion protein also displayed calcium-dependent calmodulin-binding activity in blot overlay experiments and one fusion protein bound specifically to both Drosophila and bovine brain beta-spectrin subunits on protein blots. A region of the Drosophila cDNA cross-hybridized at lowered stringency with an avian alpha-spectrin cDNA. Together these data show that the composition, structure, and binding properties of the spectrin family of proteins have been remarkably well conserved between arthropods and vertebrates. Drosophila cDNA hybridized to an mRNA of greater than or equal to 9 kb on blots of total Drosophila poly A+ RNA; and hybridized in situ to a single site in polytene region 62B, 1-7. This result and Southern blot analysis of genomic DNA indicate that the sequences are likely to be single copy in the Drosophila genome.     

Cell shape and interaction defects in alpha-spectrin mutants of Drosophila melanogaster

We show that the alpha-spectrin gene is essential for larval survival and development by characterizing several alpha-spectrin mutations in Drosophila. P-element minigene rescue and sequence analysis were used to identify the alpha-spectrin gene as the l(3)dre3 complementation group of the Dras-Roughened-ecdysoneless region of chromosome 3 (Sliter et al., 1988). Germ line transformants carrying an alpha-spectrin cDNA, whose expression is driven by the ubiquitin promoter, fully rescued the first to second instar lethality characteristic of the l(3)dre3 alleles. The molecular defects in two gamma-ray-induced alleles were identified. One of these mutations, which resulted in second instar lethality, contained a 73-bp deletion in alpha-spectrin segment 22 (starting at amino acid residue 2312), producing a premature stop codon between the two EF hands found in this segment. The second mutation, which resulted in first instar lethality, contained a 20 base pair deletion in the middle of segment 1 (at amino acid residue 92), resulting in a premature stop codon. Examination of the spectrin- deficient larvae revealed a loss of contact between epithelial cells of the gut and disruption of cell-substratum interactions. The most pronounced morphological change was seen in tissues of complex cellular architecture such as the middle midgut where a loss of cell contact between cup-shaped cuprophilic cells and neighboring interstitial cells was accompanied by disorganization of the cuprophilic cell brush borders. Our examination of spectrin deficient larvae suggests that an important role of non-erythroid spectrin is to stabilize cell to cell interactions that are critical for the maintenance of cell shape and subcellular organization within tissues.     

The complete cDNA and polypeptide sequences of human erythroid alpha-spectrin.

Overlapping human erythroid alpha-spectrin cDNA clones were isolated from lambda gt11 libraries constructed from cDNAs of human fetal liver and erythroid bone marrow. The composite 8001-base pair (bp) cDNA nucleotide sequence contains 187-bp 5'- and 528-bp 3'-untranslated regions and has a single long open reading frame of 7287 bp that encodes a polypeptide of 2429 residues. As previously described (Speicher, D. W., and Marchesi, V. T. (1984) Nature 311, 177-180), spectrin is composed largely of homologous 106-amino acid repeat units. From the amino acid sequence deduced from the cDNA, alpha-spectrin can be divided into 22 segments. Segments 1-9 and 12-19 are homologous and can therefore be considered repeats; the average number of identical residues in pairwise comparisons of these repeats is 22 out of 106, or 21%. Of these 17 repeats, 11 are exactly 106 amino acids in length, whereas five others differ from this length by a single residue. Segments 11, 20, and 21, although less homologous, appear to be related to the more highly conserved repeat units. The very N-terminal 22 residues, segment 10, which is atypical both in length and sequence, and the C-terminal 150 residues in segment 22 appear to be unrelated to the conserved repeat units. The sequence of the erythroid alpha-spectrin polypeptide chain is compared to that of human alpha-fodrin and chicken alpha-actinin to which it is related. alpha-Spectrin is more distantly related to dystrophin.     

Sequences downstream of the erythroid promoter are required for high level expression of the human alpha-spectrin gene

Alpha-spectrin is a membrane protein critical for the flexibility and stability of the erythrocyte. We are attempting to identify and characterize the molecular mechanisms controlling the erythroid-specific expression of the alpha-spectrin gene. Previously, we demonstrated that the core promoter of the human alpha-spectrin gene directed low levels of erythroid-specific expression only in the early stages of erythroid differentiation. We have now identified a region 3' of the core promoter that contains a DNase I hypersensitive site and directs high level, erythroid-specific expression in reporter gene/transfection assays. In vitro DNase I footprinting and electrophoretic mobility shift assays identified two functional GATA-1 sites in this region. Both GATA-1 sites were required for full activity, suggesting that elements binding to each site interact in a combinatorial manner. This region did not demonstrate enhancer activity in any orientation or position relative to either the alpha-spectrin core promoter or the thymidine kinase promoter in reporter gene assays. In vivo studies using chromatin immunoprecipitation assays demonstrated hyperacetylation of this region and occupancy by GATA-1 and CBP (cAMP-response element-binding protein (CREB)-binding protein). These results demonstrate that a region 3' of the alpha-spectrin core promoter contains a GATA-1-dependent positive regulatory element that is required in its proper genomic orientation. This is an excellent candidate region for mutations associated with decreased alpha-spectrin gene expression in patients with hereditary spherocytosis and hereditary pyropoikilocytosis.     

Molecular basis and haplotyping of the alphaII domain polymorphisms of spectrin: application to the study of hereditary elliptocytosis and pyropoikilocytosis.

Hereditary elliptocytosis (HE) and hereditary pyropoikilocytosis (HPP) are inherited disorders of erythrocyte shape that are frequently associated with abnormalities in alpha-spectrin, one of the principal structural proteins of the erythrocyte membrane skeleton. Five polymorphisms of the alpha-spectrin gene, located in a 6-kb interval of genomic DNA, were identified and analyzed in normal and mutant alpha-spectrin alleles. Three of these polymorphisms are due to single nucleotide substitutions in the alpha-spectrin gene coding region that lead to changes in the amino acid sequence. In combination, these three polymorphisms are responsible for the different peptide phenotypes of the alphaII domain previously observed following limited tryptic digestion of spectrin protein. The most common haplotype, type 1, was found predominantly in Caucasians and was the only haplotype identified in Asians. Haplotypes 2, 3, and 4 were identified predominantly in individuals of African ancestry and were commonly found in patients with HE or HPP. Analysis of coinheritance of alphaII domain polymorphisms with alpha-spectrin gene mutations causing HE or HPP in African-American patients with HE and HPP suggests that, with one exception, a given HE/HPP mutation is present in an alpha-spectrin gene of only one haplotype, indicating a founder effect. The other two polymorphisms located in this region of the alpha-spectrin gene do not change the amino acid sequence of the encoded alpha-spectrin chain and are not in linkage disequilibrium with three of the four alphaII domain haplotypes. A model is proposed for the evolutionary origin of the different haplotypes.     

Cloning a cDNA for the lysosomal alpha-glucosidase

Messenger RNA was isolated from monkey testes and size-fractionated on sucrose gradients. In vitro translation of these mRNA fractions resulted in nascent, labeled alpha-glucosidase that could be precipitated with anti human alpha-glucosidase antiserum. A cDNA library was constructed from the most enriched fraction. The library was screened with cDNA made from mRNA obtained from immunoselected polysomes. Five cross-hybridizing clones were isolated and identified by their selection of alpha-glucosidase mRNA, as shown by hybrid released translation and further by their ability to hybridize with DNA from human chromosome 17, on which the gene coding for acid alpha-glucosidase is located.     

From the spectrin gene to the assembly of the membrane skeleton

The complete nucleotide sequence coding for the chicken brain alpha-spectrin was determined. It comprises the entire coding frame, 5'- and 3'-untranslated sequences terminating in a poly(A)-tail. The deduced amino acid sequence shows that the alpha-chain contains 22 segments, 20 of which correspond to the typical 106 residue repeat of the human erythrocyte spectrin. Some segments non-homologous to the repeat structure reside in the middle and COOH-terminal regions. Sequence comparisons with other proteins show that these segments evidently harbour some structural and functional features such as: homology to alpha-actinin and dystrophin, two typical EF-hand structures (calcium-binding) and a putative calmodulin-binding site in the COOH-terminus and a sequence homologous to various src-tyrosine kinases and to phospholipase C in the middle of the molecule. Comparison of our sequence with other partial alpha-spectrin sequences shows that alpha-spectrin is well conserved in different species and that the human erythrocyte alpha-spectrin is divergent.     

alpha-Spectrin has a stage-specific asymmetrical localization during Xenopus oogenesis

Xenopus oocyte organization largely depends upon the cytoskeleton distribution, which is dynamically regulated during oogenesis. An actin-based cytoskeleton is present in the cortex starting from stage 1. At stages 4-6, a complex and polarized cytoskeleton network forms in the cytoplasm. In this paper, we studied the distribution of spectrin, a molecule that has binding sites for several cytoskeletal proteins and is responsible for the determination of regionalized membrane territories. The localization of alpha-spectrin mRNA was analyzed during Xenopus oogenesis by in situ hybridization on both whole mount and sections, utilizing a cDNA probe encoding a portion of Xenopus alpha-spectrin. Furthermore, an antibody against mammalian alpha-spectrin was used to localize the protein. Our results showed a stage-dependent mRNA localization and suggested that spectrin may participate in the formation of specific domains in oocytes at stages 1 and 2 and 4-6. Mol. Reprod. Dev. 55:229-239, 2000.     

Primary structure of the brain alpha-spectrin [published erratum appears in J Cell Biol 1989 Mar;108(3):following 1175]

We have determined the nucleotide sequence coding for the chicken brain alpha-spectrin. It is derived both from the cDNA and genomic sequences, comprises the entire coding frame, 5' and 3' untranslated sequences, and terminates in the poly(A)-tail. The deduced amino acid sequence was used to map the domain structure of the protein. The alpha-chain of brain spectrin contains 22 segments of which 20 correspond to the repeat of the human erythrocyte spectrin (Speicher, D. W., and V. T. Marchesi. 1984. Nature (Lond.). 311:177-180.), typically made of 106 residues. These homologous segments probably account for the flexible, rod-like structure of spectrin. Secondary structure prediction suggests predominantly alpha-helical structure for the entire chain. Parts of the primary structure are excluded from the repetitive pattern and they reside in the middle part of the sequence and in its COOH terminus. Search for homology in other proteins showed the presence of the following distinct structures in these nonrepetitive regions: (a) the COOH-terminal part of the molecule that shows homology with alpha-actinin, (b) two typical EF-hand (i.e., Ca2+-binding) structures in this region, (c) a sequence close to the EF-hand that fulfills the criteria for a calmodulin-binding site, and (d) a domain in the middle of the sequence that is homologous to a NH2-terminal segment of several src-tyrosine kinases and to a domain of phospholipase C. These regions are good candidates to carry some established as well as some yet unestablished functions of spectrin. Comparative analysis showed that alpha-spectrin is well conserved across the species boundaries from Xenopus to man, and that the human erythrocyte alpha-spectrin is divergent from the other spectrins.     

Developmental changes in the pattern of larval beta-globin gene expression in Xenopus laevis. Identification of two early larval beta-globin mRNA sequences

We have analysed beta-globin mRNA sequences in total RNA extracted from embryos and tadpoles of Xenopus laevis at different stages of development and we have identified the most abundantly transcribed beta-globin mRNA (beta T1). The entire nucleotide sequence of a cDNA clone corresponding to this mRNA is known. We have now identified the gene corresponding to this mRNA and we have determined the nucleotide sequences of its immediate 5'-flanking region. Using a DNA fragment from within the coding region of the cloned beta T1 cDNA we show, by primer extension analysis, that beta T1 mRNA is first detectable at stage 28-32 of development. This is the time at which the first presumptive erythropoietic tissue, the ventral blood island, becomes observable histologically. We show that two minor beta-globin genes, distinct from beta T1, are expressed during early stages of development, and that their expression ceases shortly after the beginning of the feeding stage. We term these two early larval genes beta E1 and beta E2. A third minor beta-globin gene is expressed during early development but, unlike beta E1 and beta E2, it is also expressed throughout subsequent larval development. We term this gene beta T2 and show that it corresponds to a gene previously termed beta LII. Finally, using a primer derived from the major adult beta-globin gene (beta 1), we have analysed the accumulation of the major adult beta-globin mRNA during larval development, and we show that this sequence does not accumulate to any significant level before metamorphosis.     

Flexibility of the alpha-spectrin N-terminus by EPR and fluorescence polarization

The structure and flexibility of the biologically important alpha-spectrin amino terminal region was examined by the use of fluorescence and EPR spectroscopy. The region studied has been previously demonstrated to be essential for the alpha-spectrin:beta-spectrin association of the tetramerization site. Appropriate spectroscopic probe moieties were coupled to this region in a recombinant fragment of human erythroid alpha-spectrin. There was good agreement between the EPR and fluorescence techniques in most of this region. Mobility determinations indicated that a portion of the region was relatively immobilized. This is significant, since although predictive methods have indicated that this region should be alpha-helical, previous experimental evidence obtained on smaller synthetic peptides had indicated that this region was disordered. Observed rigidity appears to be incompatible with such a disordered state, and has important ramifications for the flexibility of this molecule that is so integral to its role in stabilizing erythrocyte membranes.     

Drosophila development requires spectrin network formation

The head-end associations of spectrin give rise to tetramers and make it possible for the molecule to form networks. We analyzed the head-end associations of Drosophila spectrin in vitro and in vivo. Immunoprecipitation assays using protein fragments synthesized in vitro from recombinant DNA showed that interchain binding at the head end was mediated by segment 0-1 of alpha-spectrin and segment 18 of beta- spectrin. Point mutations equivalent to erythroid spectrin mutations that are responsible for human hemolytic anemias diminished Drosophila spectrin head-end interchain binding in vitro. To test the in vivo consequence of deficient head-end interchain binding, we introduced constructs expressing head-end interchain binding mutant alpha-spectrin into the Drosophila genome and tested for rescue of an alpha-spectrin null mutation. An alpha-spectrin minigene lacking the codons for head- end interchain binding failed to rescue the lethality of the null mutant, whereas a minigene with a point mutation in these codons overcame the lethality of the null mutant in a temperature-dependent manner. The rescued flies were viable and fertile at 25 degrees C, but they became sterile because of defects in oogenesis when shifted to 29 degrees C. At 29 degrees C, egg chamber tissue disruption and cell shape changes were evident, even though the mutant spectrin remained stably associated with cell membranes. Our results show that spectrin's capacity to form a network is a crucial aspect of its function in nonerythroid cells.