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.     

Proteins with spectrin motifs which do not belong to the spectrin-alpha-actinin-dystrophin family

Using several consensus sequences for the 106 amino acid residue alpha-spectrin repeat segment as probes we searched animal sequence databases using the BLAST program in order to find proteins revealing limited, but significant similarity to spectrin. Among many spectrins and proteins from the spectrin-alpha-actinin-dystrophin family as well as sequences showing a rather high degree of similarity in very short stretches, we found seven homologous animal sequences of low overall similarity to spectrin but showing the presence of one or more spectrin-repeat motifs. The homology relationship of these sequences to alpha-spectrin was further analysed using the SEMIHOM program. Depending on the probe, these segments showed the presence of 6 to 26 identical amino acid residues and a variable number of semihomologous residues. Moreover, we found six protein sequences, which contained a sequence fragment sharing the SH3 (sarc homology region 3) domain homology of 42-59% similarity. Our data indicate the occurrence of motifs of significant homology to alpha-spectrin repeat segments among animal proteins, which are not classical members of the spectrin-alpha-actinin-dystrophin family. This might indicate that these segments together with the SH3 domain motif are conserved in proteins which possibly at the early stage of evolution were close cognates of spectrin-alpha-actinin-dystrophin progenitors but then evolved separately.     

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.     

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.     

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.     

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.     

Nonallelic histone gene clusters of individual sea urchins (Lytechinus pictus): mapping of homologies in coding and spacer DNA

The linear arrangement and lengths of the spacers and coding regions in the two nonallelic histone gene variant clusters of L. pictus are remarkably homologous by R loop analysis and are similar in general topography to the histone gene repeat units of other sea urchins examined to date. No interventing sequences were detected. The coding regions of these two histone gene variants share considerable sequence homology; however, there are areas of nonhomology in every spacer region and the lengths of the nonhomologous spacers between the H2A and H1 genes are not the same for the two repeat unit classes (inter-gene heterogeneity). Combining length measurements obtained with both R loops and heteroduplexes suggests that the DNA sequences of the analogous leader regions for the two H1 mRNAs are nonhomologous. Similar observations were made for the H4 leader sequences, as well as the trailer region on H2B. S. purpuratus spacer DNA segments share little sequence homology with L. pictus; however, the analgous coding (and possibly flanking) regions have conserved their sequences. The various coding and spacer regions within a repeat unit do not share DNA sequences. Thus certain areas in the sea urchin histone gene repeat units have been highly conserved during evolution, while other areas have been allowed to undergo considerable sequence change not only between species but within a species.     

Sequence of an expressed human beta-tubulin gene containing ten Alu family members

The complete sequence of a functionally expressed human beta-tubulin gene (5 beta) is presented. The amino acid sequence encoded by this gene constitutes a distinct isotype, differing from a previously described human beta-tubulin sequence at 21 positions throughout the polypeptide chain. The beta-tubulin coding sequence in 5 beta is interrupted by three intervening sequences of 1014, 117 and 4826 nucleotides. The largest of these contains ten members of the Alu family of middle repetitive sequences. Together, these regions account for sixty percent of this intervening sequence. Two of the Alu elements are juxtaposed head to tail, and share the same flanking direct repeat. The ten Alu sequences are substantially divergent, both from each other and from an Alu consensus sequence, and several contain deletions of up to half the entire sequence.     

The beta globin gene cluster of the prosimian primate Galago crassicaudatus: nucleotide sequence determination of the 41-kb cluster and comparative sequence analyses.

The nucleotide sequence of the beta globin gene cluster of the prosimian Galago crassicaudatus has been determined. A total sequence spanning 41,101 bp contains and links together previously published sequences of the five galago beta-like globin genes (5'-epsilon-gamma-psi eta-delta-beta-3'). A computer-aided search for middle interspersed repetitive sequences identified 10 LINE (L1) elements, including a 5' truncated repeat that is orthologous to the full-length L1 element found in the human epsilon-gamma intergenic region. SINE elements that were identified included one Alu type I repeat, four Alu type II repeats, and two methionine tRNA-derived Monomer (type III) elements. Alu type II and Monomer sequences are unique to the galago genome. Structural analyses of the cluster sequence reveals that it is relatively A+T rich (about 62%) and regions with high G+C content are associated primarily with globin coding regions. Comparative analyses with the beta globin cluster sequences of human, rabbit, and mouse reveal extensive sequence homologies in their genic regions, but only human, galago, and rabbit sequences share extensive intergenic sequence homologies. Divergence analyses of aligned intergenic and flanking sequences from orthologous human, galago, and rabbit sequences show a gradation in the rate of nucleotide sequence evolution along the cluster where sequences 5' of the epsilon globin gene region show the least sequence divergence and sequences just 5' of the beta globin gene region show the greatest sequence divergence.     

Deca-satellite: A highly polymorphic satellite that joins α-satellite in the African green monkey genome

Three different cloned segments of African green monkey DNA that contain α-satellite sequences linked to a previously undescribed, distinct monkey satellite (called deca-satellite) are described here. The cloned segments were derived from a monkey DNA library in λCharon4A that was constructed to select for junctions between α-satellite and other DNA sequences.The structure of the deca-satellite and of a junction between deca-satellite and α-satellite were studied by subcloning appropriate fragments of the original cloned segments and by sequence analysis. Deca-satellite has a ten base-pair repeat unit: the consensus sequence of the repeat units is 5′ A-A-A-C-C-G-G-N-T-C. Sequences homologous to the deca-satellite are in the middle repeated class of genomic DNA. Analysis of the organization of deca-satellite sequences by digestion of total DNA with various restriction endonucleases and hybridization with a cloned deca-satellite probe revealed extensive polymorphism in the genomes of different individual monkeys but not among the tissues of one organism. These observations indicate that the arrangement of deca-satellite sequences is continually changing.An unusual α-satellite repeat unit occurs at a junction between the α-satellite and deca-satellite. It resembles the major baboon α-satellite more closely than it does monkey α-satellite and thereby provides evidence in favor of the “library” hypothesis for satellite evolution.     

The complete sequence of Drosophila alpha-spectrin: conservation of structural domains between alpha-spectrins and alpha-actinin

We report the complete sequence of Drosophila alpha-spectrin and show that it is similar to vertebrate nonerythroid spectrins. As in vertebrates, the alpha subunit consists of two large domains of repetitive sequence (segments 1-9 and 11-19) separated by a short nonrepetitive sequence (segment 10). The 106-residue repetitive segments are defined by a consensus sequence of 54 residues. Chicken alpha-spectrin (Wasenius, V.-M., M. Saraste, P. Salven, M. Eramaa, L. Holm, V.-P. Lehto. 1989. J. Cell Biol. 108:79-93) shares 50 of these consensus positions. Through comparison of spectrin and alpha-actinin sequences, we describe a second lineage of spectrin segments (20 and 21) that differs from the 106-residue segments by an 8-residue insertion and by lack of many of the consensus residues. We present a model of spectrin evolution in which the repetitive lineage of spectrin segments and the nonrepetitive lineage of segments found in spectrin and alpha-actinin arose by separate multiplication events.     

Internal Eliminated Segments (IESs) of Oxytrichidae1

ABSTRACT Internal eliminated segments (IESs) are sequences that interrupt coding and noncoding regions of germline (micronuclear) genes of ciliated protozoa. IESs are flanked by short, unique repeat sequences, which are presumably required for precise IES excision during macronuclear development. Coding and noncoding segments of genes separated by IESs are called macronuclear-destined segments, or MDSs. We have compiled the characteristics of 89 individual IESs in 12 micronuclear genes in the Oxytricha and Stylonychia genera to define the IES phenomenon precisely, a first step in determining the origin, function and significance of IESs. Although all 89 IESs among the 12 different genes are AT-rich, they show no other similarity in sequence, length, position or number. Two main types of IESs are present. IESs that separate scrambled MDSs are significantly shorter and more frequent and have longer flanking repeat sequences than IESs that intervene between nonscrambled MDSs. A comparison of the nonscrambled gene encoding β-telomere binding protein in three species of hypotrichs shows that even in the same gene IESs are not conserved in sequence, length, position, or number from species to species. A comparison of IESs in the scrambled gene encoding actin I in the three species shows that the evolutionary behavior of IESs in a scrambled gene may be more constrained. However, IESs in the scrambled actin I gene have shifted along the DNA molecule during evolution. In total, the various studies show that IESs are hypermutable in sequence and length. They insert, excise, and shift along DNA molecules more or less randomly during evolution, with no discernible function or consequences.     

Genetic analysis of the cell binding domain region of the chicken fibronectin gene

We have determined the nucleotide sequence of the cell binding domain region of the chicken fibronectin gene and analyzed it evolutionaly. We present here the complete nucleotide sequence of 4.3 kb HindIII/EcoRI segment from the clone lambda FC23 of the chicken fibronectin gene. There were five exons in this segment. When we lined up the amino acid of exons 28, 29 and 31, three alignments, known as the Type III repeat, appeared. Tetrapeptide, -RGDS-, called the cell binding domain, existed in the second repeat, coding exon 30. It was presumed that the Type III repeats were composed of two exons in the chicken gene, the same as in the rat and humans. We found repeatedly appearing amino-acid sequences such as -TIT- (three arrays in these Type III repeats) but also found one of the amino acids substituted in the tripeptide in these Type III repeats (seven arrays). We analyzed these repeats from the point of view of evolution. We used three of the nucleotide sequences (12-18 bp) coding such -TIT- repeats as a unit length for comparing the various homologies after dividing the coding region into 56 segments. The mutual homology of the divided segments to each one of three showed 53% on average. On the other hand, the mutual nucleotide homology of the Type III repeat was 44%. This suggested that the Type III repeat may have been developed by frequent duplication of small gene units.     

The exon-intron organization of the human erythrocyte alpha-spectrin gene.

The human erythrocyte alpha-spectrin gene which spans 80 kbp has been cloned from human genomic DNA as overlapping lambda recombinants. The exon-intron junctions were identified and the exons mapped. The gene is encoded by 52 exons whose sizes range from 684 bp to the smallest of 18 bp. The donor and acceptor splice site sequences match the splice site consensus sequences, with the exception of one splice site where a donor sequence begins with -GC. The size and location of exons do not correlate with the 106-amino-acid repeat, except in three locations where the surrounding codons are conserved as well. The lack of correspondence between exons and 106-amino-acid repeat is interpreted to reflect the appearance of a spectrin-like gene from a minigene early in the evolution of eukaryotes. Since current evidence indicates that introns were present in genes before the divergence of prokaryotes and eukaryotes, it is possible that the original distribution of introns within the minigene has been lost by the random deletion of introns from the spectrin gene.     

Chaperone activity and prodan binding at the self-associating domain of erythroid spectrin

Spectrin, the major constituent protein of the erythrocyte membrane skeleton, exhibits chaperone activity by preventing the irreversible aggregation of insulin at 25 degrees C and that of alcohol dehydrogenase at 50 degrees C. The dimeric spectrin and the two subunits, alpha-spectrin and beta-spectrin prevent such aggregation appreciably better, 70% in presence of dimeric spectrin at an insulin:spectrin ratio of 1:1, than that in presence of the tetramer of 25%. Our results also show that spectrin binds to denatured enzymes alpha-glucosidase and alkaline phosphatase during refolding and the reactivation yields are increased in the presence of the spectrin derivatives when compared with those refolded in their absence. The unique hydrophobic binding site on spectrin for the fluorescence probe, 6-propionyl-2-(dimethylamino)naphthalene (Prodan) has been established to localize at the self-associating domain with the binding stoichiometry of one Prodan/both dimeric and tetrameric spectrin. The other fluorescence probe, 1-anilinonaphthalene-8-sulfonic acid, does not show such specificity for spectrin, and the binding stoichiometry is between 3 and 5 1-anilinonaphthalene-8-sulfonic acid/dimeric and tetrameric spectrin, respectively. Regions in alpha- and beta-spectrins have been found to have sequence homology with known chaperone proteins. More than 50% similarities in alpha-spectrin near the N terminus with human Hsp90 and in beta-spectrin near the C terminus with human Hsp90 and Escherichia coli DnaJ have been found, indicating a potential chaperone-like sequence to be present near the self-associating domain that is formed by portions of alpha-spectrin near the N terminus and the beta-spectrin near the C terminus. There are other patches of sequences also in both the spectrin polypeptides, at the other termini as well as in the middle of the rod domain having significant homology with well known chaperone proteins.     

Cloning and sequence analysis of a cDNA encoding poly-ubiquitin in human ovarian granulosa cells

A cDNA clone, pHGR21 encoding poly-ubiquitin, was isolated from a human ovarian granulosa cDNA library. This clone contained three complete, and part of a fourth, ubiquitin coding sequence joined head to tail with no spacer sequences. Northern analysis employing a restriction fragment comprising a complete ubiquitin coding unit indicated the existence of two mRNA species of 1.1kb and 2.8kb. Sequence comparison of pHGR21 with the known two human ubiquitin genes revealed differences to the human ubiquitin-3 repeat gene but significant homology to the human ubiquitin-9 repeat gene. The untranslated 3'-region and the adjacent ubiquitin coding repeat were found to be identical to that of the human ubiquitin-9 repeat gene. The other 3 ubiquitin coding repeats were of close homology to the fourth ubiquitin coding repeat of the human ubiquitin-9 repeat gene. These findings suggest the existence of yet another human poly-ubiquitin gene.     

Cloning and partial characterization of the cDNA encoding the fox sperm protein FSA-Acr.1 with similarities to the SP-10 antigen

We have isolated and characterized a cDNA, cFSA-Acr.1, encoding a testis-specific fox sperm antigen. The antigen is located on the inner acrosomal compartment, and is expressed during spermatogenesis on the developing acrosome of round and elongating spermatids. Database searches with the deduced amino acid sequence of cFSA-Acr.1 revealed that the clone has high homology to both human and baboon sperm protein SP-10, and the mouse sperm protein, MSA-63. The region of highest homology is within the carboxyl terminus. In the middle of the open reading frame, the fox sequence shows unique sequences absent from both the human, baboon SP-10, and mouse MSA-63 sequences. In addition to cFSA-Acr.1, two other clones were also isolated from the same fox testis cDNA library, and sequence analysis shows that they may represent alternatively spliced mRNAs coding for other FSA-Acr proteins. © 1995 Wiley-Liss, Inc.     

Macronuclear and micronuclear configurations of a gene encoding the protein synthesis elongation factor EF 1α in Stylonychia lemnae

The micronuclear and macronuclear configurations of a gene encoding the protein synthesis elongation factor EF 1 alpha in the hypotrich ciliate Stylonychia lemnae were compared. The two sequences are generally colinear. The coding sequence of the micronuclear gene is, however, interrupted by a 64 bp insert flanked by a 2 bp direct repeat in a gene region which is moderately conserved among EF 1 alpha genes of different organisms. The insertion site is distinct from known intron positions in eukaryotic EF 1 alpha genes. The insert sequence shows inverted repeats at its ends and thus exhibits typical features of an internal eliminated sequence (IES). Comparison with other such sequences in the related organism Oyxtricha nova shows that the IES falls into a new group of such elements. The macronuclear gene exhibits a strikingly limited codon usage, which cannot be simply explained by the overall base composition of the DNA but probably also relates to the very high copy number of the macronuclear gene and the putative high amount of the gene product.