Intragenic duplication and divergence in the spectrin superfamily of proteins

Many structural, signaling, and adhesion molecules contain tandemly repeated amino acid motifs. The alpha-actinin/spectrin/dystrophin superfamily of F-actin-crosslinking proteins contains an array of triple alpha-helical motifs (spectrin repeats). We present here the complete sequence of the novel beta-spectrin isoform beta(Heavy)- spectrin (beta H). The sequence of beta H supports the origin of alpha- and beta-spectrins from a common ancestor, and we present a novel model for the origin of the spectrins from a homodimeric actin-crosslinking precursor. The pattern of similarity between the spectrin repeat units indicates that they have evolved by a series of nested, nonuniform duplications. Furthermore, the spectrins and dystrophins clearly have common ancestry, yet the repeat unit is of a different length in each family. Together, these observations suggest a dynamic period of increase in repeat number accompanied by homogenization within each array by concerted evolution. However, today, there is greater similarity of homologous repeats between species than there is across repeats within species, suggesting that concerted evolution ceased some time before the arthropod/vertebrate split. We propose a two-phase model for the evolution of the spectrin repeat arrays in which an initial phase of concerted evolution is subsequently retarded as each new protein becomes constrained to a specific length and the repeats diverge at the DNA level. This evolutionary model has general applicability to the origins of the many other proteins that have tandemly repeated motifs.      

Sequence, organization, and evolution of the A+T region of Drosophila melanogaster mitochondrial DNA

The long (4.6-kb) A+T region of Drosophila melanogaster mitochondrial DNA has been cloned and sequenced. The A+T region is organized in two large arrays of tandemly repeated DNA sequence elements, with nonrepetitive intervening and flanking sequences comprising only 22% of its length. The first repeat array consists of five repeats of 338-373 bp. The second consists of four intact 464-bp repeats and a fifth partial repeat of 137 bp. Three DNA sequence elements are found to be highly conserved in D. melanogaster and in several Drosophila species with short A+T regions. These include a 300-bp DNA sequence element that overlaps the DNA replication origin and two thymidylate stretches identified on opposite DNA strands. We conclude that the length heterogeneity observed in the A+T regulatory region in mitochondrial DNAs from the genus Drosophila results from the expansion (and contraction) of the number of repeated DNA sequence elements. We also propose that the 300-bp conserved DNA sequence element, in conjunction with another primary sequence determinant, perhaps the adjacent thymidylate stretch, functions in the regulation of mitochondrial DNA replication.      

Structure and chromosomal localization of DNA sequences related to ribosomal subrepeats in Vicia faba

Subrepeating sequences of 325 bp found in the ribosomal intergenic spacer (IGS) of Vicia faba and responsible for variations in the length of the polycistronic units for rRNA were isolated and used as probes for in situ hybridization. Hybridization occurs at many regions of the metaphase chromosomes besides those bearing rRNA genes, namely chromosome ends and all the heterochromatic regions revealed by enhanced fluorescence after quinacrine staining. The DNA homologous to the 325 bp repeats that does not reside in the IGS was isolated, cloned and sequenced. It is composed of tandemly arranged 336 bp elements, each comprising two highly related 168 bp sequences. This structure is very similar to that of the IGS repeats and ca. 75% nucleotide sequence identity can be observed between these and the 168 bp doublets. The most obvious difference lies in the deletion, in the former, of a 14 bp segment from one of the two related sequences. It is hypothesized that the IGS repeats are derived from the 336 bp elements and have been transposed to ribosomal cistrons from other genome fractions. The possible relations between these sequences and others with similar structural features found in other species are discussed.by D. Schweizer     

Generalized linear mixed model for segregation distortion analysis

Background

Concerted evolution refers to the pattern in which copies of multigene families show high intraspecific sequence homogeneity but high interspecific sequence diversity. Sequence homogeneity of these copies depends on relative rates of mutation and recombination, including gene conversion and unequal crossing over, between misaligned copies. The internally repetitive intergenic spacer (IGS) is located between the genes for the 28S and 18S ribosomal RNAs. To identify patterns of recombination and/or homogenization within IGS repeat arrays, and to identify regions of the IGS that are under functional constraint, we analyzed 13 complete IGS sequences from 10 individuals representing four species in the Daphnia pulex complex.

Results

Gene conversion and unequal crossing over between misaligned IGS repeats generates variation in copy number between arrays, as has been observed in previous studies. Moreover, terminal repeats are rarely involved in these events. Despite the occurrence of recombination, orthologous repeats in different species are more similar to one another than are paralogous repeats within species that diverged less than 4 million years ago. Patterns consistent with concerted evolution of these repeats were observed between species that diverged 8-10 million years ago. Sequence homogeneity varies along the IGS; the most homogeneous regions are downstream of the 28S rRNA gene and in the region containing the core promoter. The inadvertent inclusion of interspecific hybrids in our analysis uncovered evidence of both inter- and intrachromosomal recombination in the nonrepetitive regions of the IGS.

Conclusions

Our analysis of variation in ribosomal IGS from Daphnia shows that levels of homogeneity within and between species result from the interaction between rates of recombination and selective constraint. Consequently, different regions of the IGS are on substantially different evolutionary trajectories.     

Hypervariable 3′ UTR region of plant LTR-retrotransposons as a source of novel satellite repeats

The repetitive sequence PisTR-A has an unusual organization in the pea (Pisum sativum) genome, being present both as short dispersed repeats as well as long arrays of tandemly arranged satellite DNA. Cloning, sequencing and FISH analysis of both PisTR-A variants revealed that the former occurs in the genome embedded within the sequence of Ty3/gypsy-like Ogre elements, whereas the latter forms homogenized arrays of satellite repeats at several genomic loci. The Ogre elements carry the PisTR-A sequences in their 3′ untranslated region (UTR) separating the gag-pol region from the 3′ LTR. This region was found to be highly variable among pea Ogre elements, and includes a number of other tandem repeats along with or instead of PisTR-A. Bioinformatic analysis of LTR-retrotransposons mined from available plant genomic sequence data revealed that the frequent occurrence of variable tandem repeats within 3′ UTRs is a typical feature of the Tat lineage of plant retrotransposons. Comparison of these repeats to known plant satellite sequences uncovered two other instances of satellites with sequence similarity to a Tat-like retrotransposon 3′ UTR regions. These observations suggest that some retrotransposons may significantly contribute to satellite DNA evolution by generating a library of short repeat arrays that can subsequently be dispersed through the genome and eventually further amplified and homogenized into novel satellite repeats.     

Homogenization of Tandemly Repeated Nucleotide Sequences by Distance-Dependent Nucleotide Sequence Conversion

Previous work revealed that recurrent mutations (= mutation occurring more than once) in the tandemly repeated arrays present in nontranscribed spacers (NTS) of ribosomal RNA genes (rDNA) are clustered, i.e., they most frequently occur in repeats with adjacent or alternate distribution. A possible explanation is that the likelihood of heteroduplex formation, a prerequisite of gene conversion, decreases with the distance between repeats. To test this possibility, evolution of an array of 11 initially homogeneous repeats was computer simulated using three models, two assuming that the likelihood of heteroduplex formation decreases with increasing distance between the repeats and one assuming that it is constant. Patterns of mutation distribution obtained in computer simulations were compared with the distribution of mutations found in the repeated arrays in the NTS of seven rDNA clones. The patterns of mutations generated by the models assuming that the likelihood of heteroduplex formation decreases as distance between the repeats increases agreed with the patterns observed in rDNA; the patterns generated by the model assuming that the likelihood is independent of distance between repeats disagreed with the patterns observed in the rDNA clones. The topology of the heteroduplex formed between DNA in adjacent repeats predicts that the most frequently occurring conversions in the NTS repeated arrays will be shorter than the length of the repeat. The topology of the heteroduplex also predicts that if the heteroduplex leads to crossing over a circular repeat is excised. It is speculated that the circle can transpose or can be amplified via rolling circle replication and subsequently transpose. It is also shown that homogenization of the NTS repeated arrays proceeds at different rates in different species.     

A molecular and cytogenetic survey of major repeated DNA sequences in tomato (Lycopersicon esculentum)

Summary The major families of repeated DNA sequences in the genome of tomato (Lycopersicon esculentum) were isolated from a sheared DNA library. One thousand clones, representing one million base pairs, or 0.15% of the genome, were surveyed for repeated DNA sequences by hybridization to total nuclear DNA. Four major repeat classes were identified and characterized with respect to copy number, chromosomal localization by in situ hybridization, and evolution in the family Solanaceae. The most highly repeated sequence, with approximately 77000 copies, consists of a 162 bp tandemly repeated satellite DNA. This repeat is clustered at or near the telomeres of most chromosomes and also at the centromeres and interstitial sites of a few chromosomes. Another family of tandemly repeated sequences consists of the genes coding for the 45 S ribosomal RNA. The 9.1 kb repeating unit in L. esculentum was estimated to be present in approximately 2300 copies. The single locus, previously mapped using restriction fragment length polymorphisms, was shown by in situ hybridization as a very intense signal at the end of chromosome 2. The third family of repeated sequences was interspersed throughout nearly all chromosomes with an average of 133 kb between elements. The total copy number in the genome is approximately 4200. The fourth class consists of another interspersed repeat showing clustering at or near the centromeres in several chromosomes. This repeat had a copy number of approximately 2100. Sequences homologous to the 45 S ribosomal DNA showed cross-hybridization to DNA from all solanaceous species examined including potato, Datura, Petunia, tobacco and pepper. In contrast, with the exception of one class of interspersed repeats which is present in potato, all other repetitive sequences appear to be limited to the crossing-range of tomato. These results, along with those from a companion paper (Zamir and Tanksley 1988), indicate that tomato possesses few highly repetitive DNA sequences and those that do exist are evolving at a rate higher than most other genomic sequences.     

Patterns of intra- and interarray sequence variation in alpha satellite from the human X chromosome: evidence for short-range homogenization of tandemly repeated DNA sequences

A number of processes, such as sequence conversion, unequal crossingover, and molecular drive, have been postulated to explain the homogenization of tandemly repeated DNA families. To investigate the nature and extent of such processes in the alpha satellite family of centromeric DNA, we determined the nucleotide sequence of approximately 700 bp from each of 40 representative alpha satellite repeats from six sources of human X chromosomes, obtaining a total of approximately 28 kb of sequence data. Sequence divergence among the repeats examined was low, with an average pairwise difference of approximately 1%. Pairwise comparisons of all repeats indicate that the degree of similarity for those repeats in physical proximity (within approximately 15 kb) of each other is significantly greater than that for randomly located repeats, from either the same or different X chromosomes, suggesting that the mechanisms predicted to homogenize these arrays are effectively short-range in action. Analysis of individual patterns of sequence variation allows the assignment of haplotypes for five high-copy-number diagnostic positions and reveals distinct positions of equilibrium and disequilibrium within the repeat. These analyses address hypotheses about the origin of the observed patterns of variation throughout alpha satellite evolution.     

Highly condensed potato pericentromeric heterochromatin contains rDNA-related tandem repeats

The heterochromatin in eukaryotic genomes represents gene-poor regions and contains highly repetitive DNA sequences. The origin and evolution of DNA sequences in the heterochromatic regions are poorly understood. Here we report a unique class of pericentromeric heterochromatin consisting of DNA sequences highly homologous to the intergenic spacer (IGS) of the 18S.25S ribosomal RNA genes in potato. A 5.9-kb tandem repeat, named 2D8, was isolated from a diploid potato species Solanum bulbocastanum. Sequence analysis indicates that the 2D8 repeat is related to the IGS of potato rDNA. This repeat is associated with highly condensed pericentromeric heterochromatin at several hemizygous loci. The 2D8 repeat is highly variable in structure and copy number throughout the Solanum genus, suggesting that it is evolutionarily dynamic. Additional IGS-related repetitive DNA elements were also identified in the potato genome. The possible mechanism of the origin and evolution of the IGS-related repeats is discussed. We demonstrate that potato serves as an interesting model for studying repetitive DNA families because it is propagated vegetatively, thus minimizing the meiotic mechanisms that can remove novel DNA repeats.     

Highly repeated DNA sequences in birds: the structure and evolution of an abundant, tandemly repeated 190-bp DNA fragment in parrots.

Up to 6.8% of the parrot (Psittaciformes) genome consists of a tandemly repeated, 190-bp sequence (P1) located in the centromere of many if not all chromosomes. Monomer repeats from 10 different psittacine species representing four subfamilies were isolated and cloned. The intraspecific sequence variation ranged from 1.5 to 7%. The interspecific sequence variation ranged from less than 3% between two species of cockatoos to approximately 45% between cockatoos and other parrots. The monomer sequences of all 10 parrot species contained several conserved (> 90%) sequence elements at identical locations within the repeat. A comparison with tandemly repeated DNA sequences in other avian species showed that several of these conserved elements were also present at similar locations within the 184-bp repeat of the Chilean flamingo (Phoenicopterus chilensis), suggesting a great antiquity of the repeat. One of the elements was also found in the tandemly repeated sequences of the crane (Gruidae) and falcon (Falconidae) families. The data were used for the construction of a partial most parsimonious relationship that supports a regional subdivision of the Psittaciformes.     

Origin and evolution of homologous repeated sequences in the mitochondrial DNA control region of shrews

The complete mitochondrial DNA (mtDNA) control region was amplified and directly sequenced in two species of shrew, Crocidura russula and Sorex araneus (Insectivora, Mammalia). The general organization is similar to that found in other mammals: a central conserved region surrounded by two more variable domains. However, we have found in shrews the simultaneous presence of arrays of tandem repeats in potential locations where repeats tend to occur separately in other mammalian species. These locations correspond to regions which are associated with a possible interruption of the replication processes, either at the end of the three-stranded D-loop structure or toward the end of the heavy-strand replication. In the left domain the repeated sequences (R1 repeats) are 78 bp long, whereas in the right domain the repeats are 12 bp long in C. russula and 14 bp long in S. araneus (R2 repeats). Variation in the copy number of these repeated sequences results in mtDNA control region length differences. Southern blot analysis indicates that level of heteroplasmy (more than one mtDNA form within an individual) differs between species. A comparative study of the R2 repeats in 12 additional species representing three shrew subfamilies provides useful indications for the understanding of the origin and the evolution of these homologous tandemly repeated sequences. An asymmetry in the distribution of variants within the arrays, as well as the constant occurrence of shorter repeated sequences flanking only one side of the R2 arrays, could be related to asymmetry in the replication of each strand of the mtDNA molecule. The pattern of sequence and length variation within and between species, together with the capability of the arrays to form stable secondary structures, suggests that the dominant mechanism involved in the evolution of these arrays in unidirectional replication slippage.      

Evolution of Repeated Sequence Arrays in the D-Loop Region of Bat Mitochondrial DNA

Analysis of mitochondrial DNA control region sequences from 41 species of bats representing 11 families revealed that repeated sequence arrays near the tRNA-Pro gene are present in all vespertilionine bats. Across 18 species tandem repeats varied in size from 78 to 85 bp and contained two to nine repeats. Heteroplasmy ranged from 15% to 63%. Fewer repeats among heteroplasmic than homoplasmic individuals in a species with up to nine repeats indicates selection may act against long arrays. A lower limit of two repeats and more repeats among heteroplasmic than homoplasmic individuals in two species with few repeats suggests length mutations are biased. Significant regressions of heteroplasmy, θ and π, on repeat number further suggest that repeat duplication rate increases with repeat number. Comparison of vespertilionine bat consensus repeats to mammal control region sequences revealed that tandem repeats of similar size, sequence and number also occur in shrews, cats and bighorn sheep. The presence of two conserved protein-binding sequences in all repeat units indicates that convergent evolution has occurred by duplication of functional units. We speculate that D-loop region tandem repeats may provide signal redundancy and a primitive repair mechanism in the event of somatic mutations to these binding sites.