Higher-Order Organization of Subrepeats and the Evolution of Cervid Satellite I DNA

Based on sequence analyses of 17 complete centromeric DNA monomers from ten different deer species, a model is proposed for the genesis, evolution, and genomic organization of cervid satellite I DNA. All cervid satellite I DNA arose from the initial amplification of a 31-bp DNA sequence. These 31-bp subrepeats were organized in a hierarchical fashion as 0.8-kb monomers in plesiometacarpalia deer and 1-kb monomers in telemetacarpalia deer. The higher-order repeat nature of cervid centromeric satellite DNA monomers accounts for their high intragenomic and intraspecific sequence conservation. Such high intraspecific sequence conservation validates the use of a single cervid satellite I DNA monomer from each deer species for interspecific sequence comparisons to elucidate phylogenetic relationships. Also, a specific 0.18-kb tandem duplication was observed in all 1-kb monomers, implying that 1-kb cervid satellite I DNA monomers arose from an unequal crossover event between two similar 0.8-kb ancestral DNA sequences. Received: 28 May 1996 / Accepted: 24 October 1996     

Characterization of Novel Minisatellite Repeat Loci in Atlantic Salmon (Salmo salar) and Their Phylogenetic Distribution

We present here the sequence and characterization of various minisatellite-like tandem repeat loci isolated from the genome of Atlantic salmon (Salmo salar). Their diversity of sequence and lack of core motifs common to minisatellites of other species suggest the presence of numerous and previously unidentified simple sequence repeat families in this salmonid. Evidence for their ubiquity was provided by screening of a salmon genomic library. Southern blot analysis of the phylogenetic distribution of a subset of the minisatellites found one sequence to be pervasive among vertebrates, others present only in Salmoninae or Salmonidae species, and one amplified only in Atlantic salmon. There is evidence for the positioning of microsatellite and minisatellite arrays in close proximity at many loci. Furthermore, one tandem repeat appears to have been inserted into the transposase coding region of a copy of the Tc1 transposon-like element recently identified in salmonids. Received: 9 October 1996 / Accepted: 20 May 1997     

A Concertedly Evolving Region in Chironomus, Unique Within the Telomere

Chromosome terminal, complex repeats in the dipteran Chironomus pallidivittatus show rapid concerted evolution during which there is remarkably efficient homogenization of the repeat units within and between chromosome ends. It has been shown previously that gene conversion is likely to be an important component during these changes. The sequence evolution could be a result of different processes—exchanges between repeats in the tandem array as well as information transfer between units in different chromosomes—and is therefore difficult to analyze in detail. In this study the concerted evolution of a region present only once per chromosome, at the junction between the telomeric complex repeats and the subtelomeric DNA was therefore investigated in the two sibling species C. pallidivittatus and C. tentans. Material from individual microdissected chromosome ends was used, as well as clones from bulk genomic DNA. On the telomeric side of the border pronounced species-specific sequence differences were observed, the patterns being similar for clones of different origin within each species. Mutations had been transmitted efficiently between chromosomes also when adjoining, more distally localized DNA showed great differences in sequence, suggesting that gene conversion had taken place. The evolving telomeric region bordered proximally to subtelomeric DNA with high evolutionary constancy. More proximally localized, subtelomeric DNA evolved more rapidly and showed heterogeneity between species and chromosomes. Received: 24 September 1997 / Accepted: 24 November 1997     

Temporal and Spatial Occurrence of Female Chinook Salmon Carrying a Male-Specific Genetic Marker in the Columbia River Watershed

Recent declines in many chinook salmon, Oncorhynchus tshawytscha, populations within the Columbia River watershed have prompted an examination of their reproductive biology. In a previous study many female fall chinook salmon collected in 1999 from the Hanford Reach of the Columbia River tested positive for a male-specific DNA marker (OtY1) found on the Y chromosome. The purpose of this study was to determine if females testing positive for the OtY1 marker could be found in other populations of fall chinook salmon from the Columbia River, and to assess the prevalence of OtY1 incidence in different female cohorts. Post-spawned male and female fall chinook salmon from three different naturally spawning populations (Hanford Reach, Yakima River and Ives Island) and one hatchery population (Priest Rapids Hatchery) on the Columbia River were tested in 2000 and 2001 for the OtY1 marker. Among naturally spawning populations, 57.4% of the females tested positive from the Hanford Reach, 33.3% tested positive from the Yakima River, and 32.5% tested positive from Ives Island. Of the Priest Rapids Hatchery fish, 62.5% of the females tested positive, and significant differences were detected between the 1995–1996 and 1997–1998 female cohorts from this population. No significant differences were detected between any of the female cohorts from the naturally spawning populations. All male chinook salmon samples, tested positive for OtY1.     

Evolution of Tandemly Repeated Sequences: What Happens at the End of an Array?

Tandemly repeated sequences are a major component of the eukaryotic genome. Although the general characteristics of tandem repeats have been well documented, the processes involved in their origin and maintenance remain unknown. In this study, a region on the paternal sex ratio (PSR) chromosome was analyzed to investigate the mechanisms of tandem repeat evolution. The region contains a junction between a tandem array of PSR2 repeats and a copy of the retrotransposon NATE, with other dispersed repeats (putative mobile elements) on the other side of the element. Little similarity was detected between the sequence of PSR2 and the region of NATE flanking the array, indicating that the PSR2 repeat did not originate from the underlying NATE sequence. However, a short region of sequence similarity (11/15 bp) and an inverted region of sequence identity (8 bp) are present on either side of the junction. These short sequences may have facilitated nonhomologous recombination between NATE and PSR2, resulting in the formation of the junction. Adjacent to the junction, the three most terminal repeats in the PSR2 array exhibited a higher sequence divergence relative to internal repeats, which is consistent with a theoretical prediction of the unequal exchange model for tandem repeat evolution. Other NATE insertion sites were characterized which show proximity to both tandem repeats and complex DNAs containing additional dispersed repeats. An ``accretion model' is proposed to account for this association by the accumulation of mobile elements at the ends of tandem arrays and into ``islands' within arrays. Mobile elements inserting into arrays will tend to migrate into islands and to array ends, due to the turnover in the number of intervening repeats. Received: 18 August 1997 / Accepted: 18 September 1998     

Chromosome-specific alpha satellite DNA from human chromosome 1: hierarchical structure and genomic organization of a polymorphic domain spanning several hundred kilobase pairs of centromeric DNA

The human alpha satellite repetitive DNA family is organized as distinct chromosome-specific subsets localized to the centromeric region of each chromosome. Here, we report he isolation and characterization of cloned repeat units which define a hierarchical subset of alpha satellite on human chromosome 1. This subset is characterized by a 1.9-kb higher-order repeat unit which consists of 11 tandem approximately 171-bp alpha satellite monomer repeat units. The higher-order repeat unit is itself tandemly repeated, present in at least 100 copies at the centromeric region of chromosome 1. Using pulsed-field gel electrophoresis we estimate the total array length of these tandem sequences at the centromere of chromosome 1 to be several hundred kilobase pairs. Under conditions of high stringency, the higher-order repeat probe hybridizes specifically to chromosome 1 and can be used to detect several associated restriction fragment length DNA polymorphisms. As such, this probe may be useful for molecular and genetic analyses of the centromeric region of human chromosome 1.     

Similarity of Structural Features and Evolution of Satellite DNAs from Palorus subdepressus (Coleoptera) and Related Species

A novel highly abundant satellite DNA comprising 20% of the genome has been characterized in Palorus subdepressus (Insecta, Coleoptera). The 72-bp-long monomer sequence is composed of two copies of T₂A₅T octanucleotide alternating with 22-nucleotide-long elements of an inverted repeat. Phylogenetic analysis revealed clustering of monomer sequence variants into two clades. Two types of variants are prevalently organized in an alternating pattern, thus showing a tendency to generate a new complex repeating unit 144 bp in length. Fluorescent in situ hybridization revealed even distribution of the satellite in the region of pericentric heterochromatin of all 20 chromosomes. P. subdepressus satellite sequence is clearly species specific, lacking similarity even with the satellite from congeneric species P. ratzeburgii. However, on the basis of similarity in predicted tertiary structure induced by intrinsic DNA curvature and in repeat length, P. subdepressus satellite can be classified into the same group with satellites from related tenebrionid species P. ratzeburgii, Tenebrio molitor, and T. obscurus. It can be reasonably inferred that repetitive sequences of different origin evolve under constraints to adopt and conserve particular features. Obtained results suggest that the higher-order structure and repeat length, but not the nucleotide sequence itself, are maintained through evolution of these species. Received: 23 April 1997 / Accepted: 11 July 1997     

Genetic mapping of Y-chromosomal DNA markers in Pacific salmon

Sex chromosomes in fish provide an intriguing view of how sex-determination mechanisms evolve in vertebrates. Many fish species with single-factor sex-determination systems do not have cytogenetically-distinguishable sex chromosomes, suggesting that few sex-specific sequences or chromosomal rearrangements are present and that sex-chromosome evolution is thus at an early stage. We describe experiments examining the linkage arrangement of a Y-chromosomal GH pseudogene (GH-Y) sequence in four species of salmon (chum, Oncorhynchus keta; pink, O. gorbuscha; coho, O. kisutch; chinook, O. tshawytscha). Phylogenetic analysis indicates that GH-Y arose early in Oncorhynchus evolution, after this genus had diverged from Salmo and Salvelinus. However, GH-Y has not been detected in some Oncorhynchus species (O. nerka, O. mykiss and O. clarki), consistent with this locus being deleted in some lineages. GH-Y is tightly linked genetically to the sex-determination locus on the Y chromosome and, in chinook salmon, to another Y-linked DNA marker OtY1. GH-Y is derived from an ancestral GH2 gene, but this latter functional GH locus is autosomal or pseudoautosomal. YY chinook salmon are viable and fertile, indicating the Y chromosome is not deficient of vital genetic functions present on the X chromosome, consistent with sex chromosomes that are in an early stage of divergence.     

Structure and evolution of the U2 small nuclear RNA multigene family in primates: gene amplification under natural selection?

The organization of U2 genes was compared in apes, Old World monkeys, and the prosimian galago. In humans and all apes (gibbon, orangutan, gorilla, and chimpanzee), the U2 genes were organized as a tandem repeat of a 6-kb element; however, the restriction maps of the 6-kb elements in these divergent species differed slightly, demonstrating that mechanisms must exist for maintaining sequence homogeneity within this tandem array. In Old World monkeys, the U2 genes were organized as a tandem repeat of an 11-kb element; the restriction maps of the 11-kb elements in baboon and two closely related macaques, bonnet and rhesus monkeys, also differed slightly, confirming that efficient sequence homogenization is an intrinsic property of the U2 tandem array. Interestingly, the 11-kb monkey repeat unit differed from the 6-kb hominid repeat unit by a 5-kb block of monkey-specific sequence. Finally, we found that the U2 genes of the prosimian galago were dispersed rather than tandemly repeated, suggesting that the hominid and Old World monkey U2 tandem arrays resulted from independent amplifications of a common ancestral U2 gene. Alternatively, the 5-kb monkey-specific sequence could have been inserted into the 6-kb array or deleted from the 11-kb array soon after divergence of the hominid and Old World monkey lineages.     

Ribosomal DNA Intergenic Spacer of the Swimming Crab, Charybdis japonica

We have determined the full sequence of the ribosomal DNA intergenic spacer (IGS) of the swimming crab, Charybdis japonica, by long PCR for the first time in crustacean decapods. The IGS is 5376 bp long and contains two nonrepetitive regions separated by one long repetitive region, which is composed mainly of four subrepeats (subrepeats I, II, III, and IV). Subrepeat I contains nine copies of a 60-bp repeat unit, in which two similar repeat types (60 bp-a and 60 bp-b) occur alternatively. Subrepeat II consists of nine successive repeat units with a consensus sequence length of 142 bp. Subrepeat III consists of seven copies of another 60-bp repeat unit (60 bp-c) whose sequence is complementary to that of subrepeat I. Immediately downstream of subrepeat III is subrepeat IV, consisting of three copies of a 391-bp repeat unit. Based on comparative analysis among the subrepeats and repeat units, a possible evolutionary process responsible for the formation of the repetitive region is inferred, which involves the duplication of a 60-bp subrepeat unit (60 bp-c) as a prototype. Received: 13 April 1999 / Accepted: 2 August 1999     

Organizational variation of DYZ1 repeat sequences on the human Y chromosome and its diagnostic potentials

The long arm of the human Y chromosome is flecked with various fractions of repetitive DNA. DYZ1 is one such fraction, which is organized tandemly as an array of a 3.4-kb repeat ranging from 2000-4000 copies in normal males. We have studied the organizational variation of the DYZ1 fraction on the human Y chromosome using DNA samples from CEPH family members and the random population employing the RFLP approach, fluorescence in situ hybridization (FISH), and conducted a similarity search with GenBank sequences. Typing of genomic DNA using DYZ1 as a probe showed an allele length and copy number variations even between two male siblings. Hybridization of DNA from monochromosome hybrids with this probe showed its presence on chromosome 15 in addition to the Y chromosome. Fluorescence in situ hybridization of metaphase chromosomes from an apparently normal male showed DYZ1 sequences in the proximal region of chromosome 11 in addition to the long arm of the Y chromosome. Typing of sets of semen and blood DNA samples from the same human individuals showed discernible allelic variation between the two samples, indicating tissue-specific programmed sequence modulation. DYZ1 seems to be the first probe having the unique potential to discriminate unequivocally the difference between the DNA originating from semen and blood samples, and may be exploited in forensic cases. This probe may also be used as a diagnostic tool to ascertain Y chromosome mosaicism in patients (e.g., Turner), its aberrant status in somatic cells, and possible sequence modulation/rearrangement in the germline samples. Additionally, this can be used to uncover sequence polymorphism in the human population.     

Extensive Amplification and Transposition of a Novel Repetitive Element, Xstir, Together with Its Terminal Inverted Repeat in the Evolution of Xenopus

A DNA fragment containing short tandem repeat sequences (approximately 86-bp repeat) was isolated from a Xenopus laevis cDNA library. Southern blot and in situ hybridization analyses revealed that the repeat was highly dispersed in the genome and was present at approximately 1 million copies per haploid genome. We named this element Xstir (Xenopus short tandemly and invertedly repeating element) after its arrangement in the genome. The majority of the genomic Xstir sequences were digested to monomer and dimer sizes with several restriction enzymes. Their sequences were found to be highly homogeneous and organized into tandem arrays in the genome. Alignment analyses of several known sequences showed that some of the Xstir-like sequences were also organized into interspersed inverted repeats. The inverted repeats consisted of an inverted pair of two differently modified Xstirs separated by a short insert. In addition, these were framed by another novel inverted repeat (Xstir-TIR). The Xstir-TIR sequence was also found at the ends of tandem Xstir arrays. Furthermore, we found that Xstir-TIR was linked to a motif characterizing the T2 family which belonged to a vertebrate MITE (miniature inverted-repeat transposable element) family, suggesting the importance of Xstir-TIR for their amplification and transposition. The present study of 11 anuran and 2 urodele species revealed that Xstir or Xstir-like sequences were extensively amplified in the three Xenopus species. Genomic Xstir populations of X. borealis and X. laevis were mutually indistinguishable but significantly different from that of X. tropicalis. Received: 5 April 2000 / Accepted: 3 August 2000     

Population Genetics of Y-Chromosome Short Tandem Repeats in Humans

Eight human short tandem repeat polymorphisms (STRs) also known as microsatellites—DYS19, DYS388, DYS390, DYS391, DYS392, DYS393, DYS389I, and DYS389II, mapping in the Y chromosome—were analyzed in two Iberian samples (Basques and Catalans). Allele frequency distributions showed significant differences only for DYS392. Fst and gene diversity index (D) were estimated for the Y STRs. The values obtained are comparable to those of autosomal STR if corrections for the smaller effective population size on the Y chromosome are taken into account. This suggests that Y-chromosome microsatellites might be as useful as their autosomal counterparts to both human population genetics and forensics. Our results also reinforce the hypothesis that selective sweeps in the Y chromosome in recent times are unlikely. Haplotypes combining five of the loci were constructed for 71 individuals, showing 29 different haplotypes. A haplotype tree was constructed, from which an estimate of 7,000 to 60,000 years for the age of the Y-chromosome variation in Iberia was derived, in accordance with previous estimates obtained with mtDNA sequences and nuclear markers. Received: 3 January 1997 / Accepted: 25 April 1997     

Chromosome-specific alpha satellite DNA from the centromere of human chromosome 16

To examine the molecular organization of DNA sequences located in the centromeric region of human chromosome 16 we have isolated and characterized a chromosome 16-specific member of the alpha satellite DNA family. The probe obtained is specific for the centromere of chromosome 16 by somatic cell hybrid analysis and by fluorescence in situ hybridization and allows detection of specific hybridizing domains in interphase nuclei. Nucleotide sequence analysis indicates that this class of chromosome 16 alpha satellite (D16Z2) is organized as a series of diverged 340-bp dimers arranged in a tandem array of 1.7-kb higher-order repeat units. As measured by pulsed-field gel electrophoresis, the total D16Z2 array spans approximately 1,400-2,000 kb of centromeric DNA. These sequences are highly polymorphic, both by conventional agarose-gel electrophoresis and by pulsed-field gel electrophoresis. Investigation of this family of alpha satellite should facilitate the further genomic, cytogenetic, and genetic analysis of chromosome 16.     

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.     

Characterization of the nuclear ribosomal DNA of Euglena gracilis

A phage lambda recombinant library containing Euglena gracilis genomic DNA was screened for nuclear rDNA sequences. A recombinant phage was isolated that contained an 11.5-kb nuclear rDNA sequence. The 11.5-kb insert was mapped with restriction endonucleases and was shown to represent a complete rDNA repeat unit that carried the genes for the 19S, 25S, 5.8 S and 5 S cytoplasmic rRNAs. The 2000 rDNA repeat units per haploid genome are organized in the form of identical tandem repeats.     

Tandem repeat DNA localizing on the proximal DAPI bands of chromosomes in Larix, Pinaceae.

Repetitive DNA was cloned from HindIII-digested genomic DNA of Larix leptolepis. The repetitive DNA was about 170 bp long, had an AT content of 67%, and was organized tandemly in the genome. Using fluorescence in situ hybridization and subsequent DAPI banding, the repetitive DNA was localized in DAPI bands at the proximal region of one arm of chromosomes in L. leptolepis and Larix chinensis. Southern blot hybridization to genomic DNA of seven species and five varieties probed with cloned repetitive DNA showed that the repetitive DNA family was present in a tandem organization in genomes of all Larix taxa examined. In addition to the 170-bp sequence, a 220-bp sequence belonging to the same DNA family was also present in 10 taxa. The 220-bp repeat unit was a partial duplication of the 170-bp repeat unit. The 220-bp repeat unit was more abundant in L. chinensis and Larix potaninii var. macrocarpa than in other taxa. The repetitive DNA composed 2.0-3.4% of the genome in most taxa and 0.3 and 0.5% of the genome in L. chinensis and L. potaninii var. macrocarpa, respectively. The unique distribution of the 220-bp repeat unit in Larix indicates the close relationship of these two species. In the family Pinaceae, the LPD (Larix proximal DAPI band specific repeat sequence family) family sequence is widely distributed, but their amount is very small except in the genus Larix. The abundant LPD family in Larix will occur after its speciation.