Extensive Direct-Tandem Organization of a Long Repeat DNA Sequence on the Y Chromosome of Chinook Salmon (Oncorhynchus tshawytscha)

A long repetitive DNA sequence (OtY8) has been cloned from male chinook salmon and its genomic organization has been characterized. The repeat has a unit length of 8 kb and is present approximately 300 times per diploid male nucleus. All internal fragments within the 8-kb repeat segregate from father to son, suggesting that the entire repeat unit is located on the Y chromosome. The organization of this sequence into an 8-kb repeat unit is restricted to the Y chromosome, as are several male-specific repeat subtypes identified on the basis of restriction-site variation. The repeat possesses only weak internal sequence similarities, suggesting that OtY8 has not arisen by duplication of a smaller repeat unit, as is the case for other long tandem arrays found in eukaryotes. Based on a laddered pattern arising from partial digestion of genomic DNA with a restriction enzyme which cuts only once per repeat unit, this sequence is not dispersed on the Y chromosome but is organized as a head-to-tail tandem array. Pulse-gel electrophoresis reveals that the direct-tandem repeats are organized into at least six separate clusters containing approximately 12 to 250 copies, comprising some 2.4 Mb of Y-chromosomal DNA in total. Related sequences with nucleotide substitutions and DNA insertions relative to the Y-chromosomal fragment are found elsewhere in the genome but at much lower copy number and, although similar sequences are also found in other salmonid species, the amplification of the repeat into a Y-chromosome-linked tandem array is only observed in chinook salmon. The OtY8 repetitive sequence is genetically tightly associated with the sex-determination locus and provides an opportunity to examine the evolution of the Y chromosome and sex determination process in a lower vertebrate. Received: 4 April 1997 / Accepted: 22 July 1997     

Nucleotide sequence, genomic organization and evolution of a major repetitive DNA family in tilapia (Oreochromis mossambicus/hornorum).

A highly repetitive DNA sequence from tilapia (Oreochromis mossambicus/hornorum) has been cloned and sequenced. It is a tandemly arrayed sequence of 237 bp and constitutes 7% of the fish genome. The copy number of the repeat is approximately 3 x 10(5) per haploid genome. DNA sequence analysis of 7 cloned repeats revealed a high degree of conservation of the monomeric unit. Within the monomeric unit, a 9 bp AT rich motif is regularly spaced approximately 30 bp apart and may represent the progenitor of the amplified sequence. One cloned repeat, Ti-14, contained a 30 bp deletion at a position flanked by a 7 bp direct repeat. The Ti-14 sequence appears to have been amplified independently of the major 237 bp tandem array. A higher-order repeat unit, defined by longer-range periodicities revealed by restriction endonuclease digestion, is further imposed on the tandem array.     

Characterization of a porcine variable number tandem repeat sequence specific for the glucosephosphate isomerase locus

A variable number of tandem repeat from a porcine glucosephosphate isomerase intron has been isolated and sequenced. The repeat has a unit size of 39 bp, is highly conserved and is present in at least 14 copies. Flanking sequences show a sequence periodicity of 53-54 bp and some sequence homology to the 39 bp repeat. A considerable part of the genomic DNA has been lost during subcloning and is considered to be deletion prone or refractory to propagation in E. coli. The tandem repeat is locus specific and detects at least six alleles in BamHI digested porcine DNA. No homology to other tandem repeat sequences has been found.     

Molecular analysis of a polymorphic domain of alpha satellite from the human X chromosome.

Alpha satellite DNA, a diverse family of tandemly repeated DNA sequences located at the centromeric region of each human chromosome, is organized in a highly chromosome-specific manner and is characterized by a high frequency of restriction-fragment-length polymorphism. To examine events underlying the formation and spread of these polymorphisms within a tandem array, we have cloned and sequenced a representative copy of a polymorphic array from the X chromosome and compared this polymorphic copy with the predominant higher-order repeat form of X-linked alpha satellite. Sequence data indicate that the polymorphism arose by a single base mutation that created a new restriction site (for HindIII) in the sequence of the predominant repeat unit. This variant repeat unit, marked by the new HindIII site, was subsequently amplified in copy number to create a polymorphic domain consisting of approximately 500 copies of the variant repeat unit within the X-linked array of alpha satellite. We propose that a series of intrachromosomal recombination events between misaligned tandem arrays, involving multiple rounds of either unequal crossing-over or sequence conversion, facilitated the spread and fixation of this variant HindIII repeat unit.     

A member of a new repeated sequence family which is conserved throughout eucaryotic evolution is found between the human delta and beta globin genes

A new class of human interspersed repeated sequences distinct from the AluI family was found by screening a human gene library with a mouse ribosomal gene non-transcribed spacer probe (rDNA NTS). A member of this sequence family was localized to a 251 bp segment between the human delta and beta globin genes: a region previously judged to be devoid of repeated DNA. The complete nucleotide sequence of this segment revealed a tandem block of 17 TG dinucleotides, a feature hypothesized by others to be a recombination hot spot responsible for gene conversion in the gamma globin locus region. When the genomes of Xenopus, pigeon, slime mold and yeast were examined, reiterated sequences homologous to both the mouse rDNA NTS and human globin repeat were found in every case. The discovery of this extraordinarily conserved repeated sequence family appears to have depended upon not using salmon sperm DNA during hybridization. The use of eucaryotic carrier DNA may bias the search for repeated sequences against any which may be highly conserved during eucaryotic evolution.     

C1 inhibitor gene sequence facilitates frameshift mutations.

Mutations disrupting the function or production of C1 inhibitor cause the disease hereditary angioneurotic edema. Patient mutations identified an imperfect inverted repeat sequence that was postulated to play a mechanistic role in the mutations. To test this hypothesis, the inverted repeat was cloned into the chloramphenicol acetyltransferase gene in pBR325 and its mutation rate was studied in four bacterial strains. These strains were selected to assay the effects of recombination and superhelical tension on mutation frequency. Mutations that revert bacteria to chloramphenicol resistance (Cmr) were scored. Both pairs of isogenic strains had reversion frequencies of approximately 10(-8). These rare reversion events in bacteria were most often a frameshift that involved the imperfect inverted repeat with a deletion or a tandem duplication, an event very similar to the human mutations. Increased DNA superhelical tension, which would be expected to enhance cruciform extrusion, did not accentuate mutagenesis. This finding suggests that the imperfect inverted repeat may form a stem-loop structure in the single-stranded DNA created by the duplex DNA melting prior to replication. Models explaining the slippage can be drawn using the lagging strand of the replication fork. In this model, the formation of a stem-loop structure is responsible for bringing the end of the deletion or duplication into close proximity.     

DNA tertiary structures formed in vitro by misaligned hybridization of multiple tandem repeat sequences.

DNA tertiary structures are shown to be formed by denaturation and reannealing in vitro of molecularly-cloned DNA containing multiple tandem repeat sequences. Electron microscopy of homoduplex DNA molecules containing the human c-Harvey-ras gene revealed knot-like structures which mapped to the position of the 812 bp variable tandem repeat (VTR) sequence. We propose that the structures result from slipped-strand mispairing within the VTR and hybridisation of homologous repetitive sequences in the single-stranded loops so produced. Similar structures were also found in freshly-linearized supercoiled plasmids. More complex knot-like structures were found in homoduplexes of a 4 kb tandem array from the hypervariable region 3' to the human alpha-globin locus. Formation of such DNA tertiary structures in vitro also provides a practical method for identifying and mapping direct tandem repeat arrays that are at least 800 bp long.     

Evolutionary rate variation among vertebrate beta globin genes: implications for dating gene family duplication events

A comprehensive dataset of 62 beta globin gene sequences from various vertebrates was compiled to test the molecular clock and to estimate dates of gene duplications. We found that evolution of the beta globin family of genes is not clock-like, a result that is at odds with the common use of this family as an example of a constant rate of evolution over time. Divergence dates were estimated either with or without assuming the molecular clock, and both analyses produced similar date estimates, which are also in general agreement with estimates reported previously. In addition we report date estimates for seven previously unexamined duplication events within the beta globin family. Despite multiple sources of rate variation, the average rate across the beta globin phylogeny yielded reasonable estimates of divergence dates in most cases. Exceptions were cases of gene conversion, where it appears to have led to underestimates of divergence dates. Our results suggest (i) the major duplications giving rise to the paralogous beta globin genes are associated with significant evolutionary rate variation among gene lineages; and (ii) genes arising from more recent gene duplications (e.g., tandem duplications within lineages) do not appear to differ greatly in rate. We believe this pattern reflects a complex interplay of evolutionary forces where natural selection for diversifying paralogous functions and lineage-specific effects contribute to rate variation on a long-term basis, while gene conversion tends to increase sequence similarity. Gene conversion effects appear to be stronger on recent gene duplicates, as their sequences are highly similar. Lastly, phylogenetic analyses do not support a previous report that avian globins are members of a relic lineage of omega globins.     

Isolation of novel herpes simplex virus type 1 derivatives with tandem duplications of DNA sequences encoding immediate-early mRNA-5 and an origin of replication.

Two naturally occurring variations of herpes simplex virus type 1 (Patton strain) with novel tandem DNA sequence duplications in the S component were isolated, and the DNA was characterized. These variants were identified among a number of plaque isolates by the appearance of new restriction enzyme fragments that hybridized with radiolabeled DNA from the BamHI Z fragment (map coordinates 0.936 to 0.949) located in the unique S region. One isolate, SP26-3, carried a 3.1-kilobase-pair duplication defined by recombination between a site in the BamHI Z fragment and a site near the origin of replication in the inverted repeat sequence of the S component carried by the EcoRI H fragment. The other isolate, SP22-4, carried a 3.5-kilobase-pair duplication defined by a recombination event between a tandem repeat array in the BamHI Z fragment and a site near the amino terminus of the Vmw175 gene in the S-region inverted repeat sequence contained in the EcoRI K fragment. Both duplicated segments contained the entire immediate early mRNA-5 coding region as well as the origin of replication located in the inverted repeat sequence of the S component. The DNA sequence of each duplication joint was determined.     

A variable tandem repeat locus mapped to chromosome band 10q26 is amplified and rearranged in leukocyte DNAs of two cancer patients.

A highly polymorphic locus associated with the variable tandem repetition of a 35 bp consensus sequence was mapped to chromosome 10, band q26. Examination of leukocyte DNA from a cancer patient revealed the twenty-fold amplification of one allelic fragment of this locus, while the other allelic fragment demonstrated a normal copy number. In another patient, Southern blotting of leukocyte DNA detected the deletion of the 3'-flanking region from one tandem repeat allele. These results indicate that variable tandem repeats may mark highly unstable regions of DNA in the human genome which can be altered by changes more extensive than simple tandem repeat variation.     

Comparison of minisatellites.

In the class of repeated sequences that occur in DNA, minisatellites have been found polymorphic and became useful tools in genetic mapping and forensic studies. They consist of a heterogeneous tandem array of a short repeat unit. The slightly different units along the array are called variants. Minisatellites evolve mainly through tandem duplications and tandem deletions of variants. Jeffreys et al. (1997) devised a method to obtain the sequence of variants along the array in a digital code and called such sequences maps. Minisatellite maps give access to the detail of mutation processes at work on such loci. In this paper, we design an algorithm to compare two maps under an evolutionary model that includes deletion, insertion, mutation, tandem duplication, and tandem deletion of a variant. Our method computes an optimal alignment in reasonable time; and the alignment score, i.e., the weighted sum of its elementary operations, is a distance metric between maps. The main difficulty is that the optimal sequence of operations depends on the order in which they are applied to the map. Taking the maps of the minisatellite MSY1 of 609 men, we computed all pairwise distances and reconstructed an evolutionary tree of these individuals. MSY1 (DYF155S1) is a hypervariable locus on the Y chromosome. In our tree, the populations of some haplogroups are monophyletic, showing that one can decipher a microevolutionary signal using minisatellite maps comparison.     

Influences of array size and homogeneity on minisatellite mutation.

Unstable minisatellites display high frequencies of spontaneous gain and loss of repeats in the human germline. Most length changes arise through complex recombination events including intra-allelic duplications/deletions and inter-allelic transfers of repeats. Definition of the factors modulating instability requires both measurement of mutation rate and detailed analysis of mutant structures at the level of individual alleles. We have measured mutation rates in sperm for a wide range of alleles of the highly unstable human minisatellite CEB1. Instability varies by three orders of magnitude between alleles and increases steadily with the size of the tandem array. Structural analysis of mutant molecules derived from six alleles revealed that it is the rate of intra-allelic rearrangements which increases with array size and that intra-allelic duplication events tend to cluster within homogeneous segments of alleles; both phenomena resemble features of trinucleotide repeat instability. In contrast, inter-allelic transfers occur at a fairly constant rate, irrespective of array length, and show a mild polarity towards one end of the minisatellite, suggesting the possible influence of flanking DNA on these conversion-like events.     

Tandemly repeated transgenes of the human minisatellite MS32 (D1S8), with novel mouse gamma satellite integration.

The human hypervariable minisatellite MS32 has a well characterised internal repeat unit array and high mutation rates have been observed at this locus. Analysis of MS32 mutants has shown that male germline mutations are polarised to one end of the array and frequently involve complex gene conversion-like events, suggesting that tandem repeat instability may be modulated by cis-acting sequences flanking the locus. In order to investigate the processes affecting MS32 mutation rate and mechanism, we have created transgenic mice harbouring an MS32 allele. Here we describe the organisation of eight transgenic insertions. Analysis of these transgenic loci by MVR-PCR and structural analysis of the junctions between mouse flanking DNA and the transgenic loci has shed light on mechanisms of integration and rearrangement of the tandem repeated transgenes. Sequence analysis of the mouse DNA flanking these transgenes has shown that 5 of the 8 insertions have integrated into mouse gamma satellite repeated sequence. This suggests a non-random integration of the MS32 transgene construct into the mouse genome.     

Concerted evolution of the tandem array encoding primate U2 snRNA occurs in situ, without changing the cytological context of the RNU2 locus.

In primates, the tandemly repeated genes encoding U2 small nuclear RNA evolve concertedly, i.e. the sequence of the U2 repeat unit is essentially homogeneous within each species but differs somewhat between species. Using chromosome painting and the NGFR gene as an outside marker, we show that the U2 tandem array (RNU2) has remained at the same chromosomal locus (equivalent to human 17q21) through multiple speciation events over > 35 million years leading to the Old World monkey and hominoid lineages. The data suggest that the U2 tandem repeat, once established in the primate lineage, contained sequence elements favoring perpetuation and concerted evolution of the array in situ, despite a pericentric inversion in chimpanzee, a reciprocal translocation in gorilla and a paracentric inversion in orang utan. Comparison of the 11 kb U2 repeat unit found in baboon and other Old World monkeys with the 6 kb U2 repeat unit in humans and other hominids revealed that an ancestral U2 repeat unit was expanded by insertion of a 5 kb retrovirus bearing 1 kb long terminal repeats (LTRs). Subsequent excision of the provirus by homologous recombination between the LTRs generated a 6 kb U2 repeat unit containing a solo LTR. Remarkably, both junctions between the human U2 tandem array and flanking chromosomal DNA at 17q21 fall within the solo LTR sequence, suggesting a role for the LTR in the origin or maintenance of the primate U2 array.     

Molecular characterization of a repeat element causing large-scale size variation in the mitochondrial DNA of the sea scallop Placopecten magellanicus

The scallop Placopecten magellanicus has the largest reported animal mitochondrial DNA (average 35 kb) and exhibits large inter- and intraindividual length variation owing to the varying copy number of a repeated element. We have characterized the repeat array by using restriction mapping and sequence analysis. The repeated element consists of 1,442 bp flanked on either side by the sequence ACTTTCC in a direct orientation. The array contains two to eight copies of the repeated element arranged in a direct orientation and in tandem. Only complete copies of the element are present in the array. The repeat element contains three regions with characteristic nucleotide sequences: a 10-bp inverted repeat shown to extrude into a cruciform in a supercoiled DNA plasmid, a 120-bp tract rich in G/C (70%) and adjacent to the inverted repeat, and periodically interspersed homopolymer runs of A and T occurring near the middle of the element which induce DNA curvature in dimeric constructs of the element. The element appears to be unique to P. magellanicus. The structural properties of the repeat element and its organization in an array of repeats may be important in explaining the generation and maintenance of large-scale mitochondrial DNA size variation observed in many animal species.     

Inversions with Deletions and Duplications

Complex mutational events, including de novo inversion with deletion and duplication of sequence, have been observed but are difficult to model. We propose that nascent leading-strand misalignment upon the lagging-strand template during DNA replication can result in the inversion of sequence. The positioning of this misalignment and of the realignment of the leading strand back onto the leading-strand template will determine if the inversion is accompanied by deletion and duplication of sequence. We suggest that such strand misalignment-realignment events may occur at the replication fork during concurrent DNA replication.     

Physical mapping of the globin gene deletion in hereditary persistence of foetal haemoglobin (HPFH).

We have mapped the globin gene region in the DNA of two HPFH patients. In a patient homozygous for the G gamma A gamma type of HPFH at least 24 kb of DNA in the globin gene region has been deleted to remove most of the gamma-delta intergenic region and the delta and beta globin genes. The 5' break point of the deletion is located about 9 kb upstream from the delta globin gene. The 3' break point has not been precisely located but is at least 7 kb past the beta globin gene. DNA from an individual heterozygous for the Greek (A gamma) type of HPFH, however, shows no detectable deletion in the entire gamma delta beta-globin gene region. HPFH, therefore, appears to occur in different molecular forms. These results are discussed in terms of a model for the regulation of globin gene expression in man.     

Length Variation in Mitochondrial DNA of the Minnow Cyprinella Spiloptera

Length differences in animal mitochondrial DNA (mtDNA) are common, frequently due to variation in copy number of direct tandem duplications. While such duplications appear to form without great difficulty in some taxonomic groups, they appear to be relatively short-lived, as typical duplication products are geographically restricted within species and infrequently shared among species. To better understand such length variation, we have studied a tandem and direct duplication of approximately 260 bp in the control region of the cyprinid fish, Cyprinella spiloptera. Restriction site analysis of 38 individuals was used to characterize population structure and the distribution of variation in repeat copy number. This revealed two length variants, including individuals with two or three copies of the repeat, and little geographic structure among populations. No standard length (single copy) genomes were found and heteroplasmy, a common feature of length variation in other taxa, was absent. Nucleotide sequence of tandem duplications and flanking regions localized duplication junctions in the phenylalanine tRNA and near the origin of replication. The locations of these junctions and the stability of folded repeat copies support the hypothesized importance of secondary structures in models of duplication formation.