Organization and evolution of highly repeated satellite DNA sequences in plant chromosomes

A major component of the plant nuclear genome is constituted by different classes of repetitive DNA sequences. The structural, functional and evolutionary aspects of the satellite repetitive DNA families, and their organization in the chromosomes is reviewed. The tandem satellite DNA sequences exhibit characteristic chromosomal locations, usually at subtelomeric and centromeric regions. The repetitive DNA family(ies) may be widely distributed in a taxonomic family or a genus, or may be specific for a species, genome or even a chromosome. They may acquire large-scale variations in their sequence and copy number over an evolutionary time-scale. These features have formed the basis of extensive utilization of repetitive sequences for taxonomic and phylogenetic studies. Hybrid polyploids have especially proven to be excellent models for studying the evolution of repetitive DNA sequences. Recent studies explicitly show that some repetitive DNA families localized at the telomeres and centromeres have acquired important structural and functional significance. The repetitive elements are under different evolutionary constraints as compared to the genes. Satellite DNA families are thought to arise de novo as a consequence of molecular mechanisms such as unequal crossing over, rolling circle amplification, replication slippage and mutation that constitute "molecular drive".     

Organization of highly repeated sequences in surface-spread pachytene chromosomes of rye.

A method of preparing two-dimensional surface spreads of plant synaptonemal complexes (SCs) associated with fluorescence in situ hybridization (FISH) has been applied to analyze the location and organization of five different highly repeated DNA sequences in rye. Our observations indicate that, depending on the type of sequence, the chromatin displays different types of organization. Telomeric sequences were seen tightly associated with the SC while other repetitive DNA sequences were found to form loops that are associated with SCs only at their bases. On the contrary, the FISH signal of a centromeric satellite had a granular appearance, reflecting that the hybridization occurs only with parts of the chromatin loops.     

Organisation and properties of repeated DNA sequences in rice genome

Reassociation of high molecular weight rice DNA has revealed the occurrence of long stretches of repeated DNA which are not interrupted by single copy DNA even at a fragment length as high as 20 kilo base pairs (kbp). Majority of these repeated sequences are unusually G + C rich and show significant variations in their thermal stability. Homology studies indicate that short repeats may have evolved from long repeats in total repetitive DNA while they may be of different origin in highly repetitive DNA fraction. Restriction enzyme analysis shows the occurrence of Ava I and EcoR V repeat families.     

Involvement of transposition in dispersion of tandem repeat sequences (TrsA) in rice genomes

We describe a method to identify and characterize DNA fragments containing the junction of AA genome-specific tandem repeat sequences (here called TrsA) with adjacent chromosomal sequences of rice by the polymerase chain reaction (PCR) using a pair of primers that hybridize with TrsAs and a flanking non-TrsA sequence. With this method, we obtained results suggesting that TrsA sequences present at two loci (here called trsA1 and trsA2) are flanked by direct repeats of chromosomal sequences of 172 by and about 440 by in length, respectively. These results support the idea that the TrsA sequences have been inserted into each locus by transposition, resulting in duplication of the chromosomal sequence used as target. We also describe a method to identify and characterize TrsA sequences repeated in only a few copies in the rice genome by PCR, using a pair of primers that hybridize with two different portions in the TrsA sequence, and demonstrate that TrsA sequences are present not only in rice strains with the AA genome, but also in those with non-AA genomes. The TrsA sequences were present at the trsA1 locus in all the rice strains examined, indicating that TrsA was inserted and amplified at the locus before the divergence of the various species of rice in the Oryza genus. TrsA sequences were present at the trsA2 locus, however, only in an O. sativa IR36 strain, indicating that TrsA was inserted and amplified at this locus during divergence of rice strains with the AA genome.     

Differential expansion of highly repeated DNA sequences in the swine subgenomes

Differences in highly repeated DNA sequences among three swine breeds genomes were detected by means of whole‐comparative genomic hybridization (W‐CGH). The results showed that Duroc, Iberian and Landrace/Large White breeds share similar DNA sequences in their centromeric regions, but the number of copies of the highly repeated DNA sequences building the blocks of heterochromatin in the metacentric chromosomes is differentially expanded among them. That is not the case in the acrocentric subgenome where the chromosomes share similar sequence composition and number of copies among the three breeds in the centromeric regions. The highly repeated DNA sequences in the chromosome Y also displayed differences among the breeds studied. The reported results are discussed in the light of the possible evolutionary tendencies of these particular DNA sequences.     

A BamHI family of highly repeated DNA sequences of Nicotiana tabacum

Summary HRS60.1, a monomer unit (184 bp) of a highly repeated nuclear DNA sequence of Nicotiana tabacum, has been cloned and sequenced. Following BamHI digestion of tobacco DNA, Southern hybridization with HRS60.1 revealed a ladder of hybridization bands corresponding to multiples of the basic monomer unit. If the tobacco DNA was digested with restriction endonucleases which have no target site in HRS60.1, the larger part of DNA homologous to HRS60.1 remained as uncleaved relic DNA. These results suggest a tandem arrangement of this DNA repeat unit. Four other clones of tobacco nuclear DNA cross-hybridized with HRS60.1, thus forming a HRS60-family. Sequencing their inserts has shown their strong mutual homology. HRS60-family comprised about 2% of the nuclear genome of N. tabacum. Computer comparisons with other tandem plant-repeated DNA sequences could not detect any other homologous sequence.     

The 5-methylcytosine content of highly repeated sequences in human DNA.

Previously, we found much tissue- or cell-specificity in the levels of 5-methylcytosine (m5C) in the total human genome as well as in DNA fractions resolved by reassociation kinetics. We now report that there were even greater differences in the m5C content of the highly repeated, tandem EcoRI family of DNA sequences from different human organs or cell populations. The ratio of m5C levels in this DNA fraction from brain, placenta, and sperm was 2.0:1.2:1.0. At a HhaI site in this repeat family, sperm DNA was 5-10 fold less methylated than somatic DNAs. In contrast, the highly repeated Alu family, which is approximately 5% of the genome, had almost the same high m5C content in brain and placenta despite marked tissue-specific differences in m5C levels of the single copy sequences with which these repeats are interspersed. These data show that very different degrees of change in methylation levels of various highly repeated DNA sequences accompany differentiation.     

Heterochromatin and highly repeated DNA sequences in rye (Secale cereale)

Secale cereale DNA, of mean fragment length 500 bp, was fractionated by hydroxylapatite chromatography to allow recovery of a very rapidly renaturing fraction (C₀t 0–0.02). This DNA fraction was shown to contain several families of highly repeated sequence DNA. Two highly repeated families were purified; (1) a fraction which renatured to a density of 1.701 g/ cc and comprised 2–4% of the total genome, and (2) polypyrimidine tract DNA which comprised 0.1% of the total genome. The 1.701 g/cc DNA consisted of short sequence repeat units (5–50 bp long) tandemly repeated in blocks 30 kb long, while a portion of the polypyrimidine tract DNA behaved as part of a much larger block of tandemly repeated sequences. The chromosomal location of these sequences was determined by the in situ hybridisation of radioactive, complementary RNA to root tip mitotic chromosomes and showed the 1.701 g/cc sequences to be largely limited to the telomeric blocks of heterochromatin, accounting for 25–50% of the DNA present in these parts of the chromosomes. The polypyrimidine tracts were distributed at interstitial locations with 20–30% of the sequences at three well defined sites. The combined distributions of the 1.701 g/cc DNA sequences and polypyrimidine tracts effectively individualised each rye chromosome thus providing a sensitive means of identifying these chromosomes. The B chromosomes present in Secale cereale cv. Unevita, did not show defined locations for the sequences analysed. — The data are discussed in terms of the structure of the rye genome and the generality of the observed genomic arrangement of highly repeated sequence DNA.     

Analysis of highly repeated DNA sequences of rat with EcoR1 endonuclease

Cleavage of rat liver nuclear DNA with EcolR1 restriction endonuclease yields 14 discrete fragments ranging from 2300 to 93 base pairs in length, representing approx. 10.5% of the rat genome. Fragments of 1500, 180, and 93 base pairs are reiterated over 100 000 times; fragments of 2300, 880, 290, and 200 base pairs are reiterated over 20 000 times; the remaining fragments are present in over 1000 copies per genome. When compared to whole rate DNA, 11 were 1-5% richer in A . T base pairs and five were 1.5-2.5 times more methylated. From the criteria of the banding patterns in complete and incomplete digests, base composition and extent of methylation, none of these fragments appeared to be generated as oligomers of a basic shorter repeat. The reassociation of EcoR1 fragments was monitored on hydroxyapatite and by S1 nuclease treatment in order to assess band reiteration frequency and the possibility of interpersion or short internal repeats. The renaturation of the four smallest EcoR1 fragments gave no indication of short internal repeats from hyperpolymer formation nor interpersion with lower frequency sequences by size reduction after S1 nuclease treatment. Anomalous renaturation of several large fragments was observed, possibly due to internal repeats.     

The distribution of two highly repeated DNA sequences within Drosophila melanogaster chromosomes

In situ hybridization using ³H-RNA probes has been used to localize the sequences found in two satellites of density 1.705 g/cc and 1.672 g/ cc to specific sites within the chromosomal complement. A detailed analysis of the sites on the X chromosome was carried out using the scute series of inversions to relate the heterochromatic breakpoint relative to the location of the sequence on this chromosome. It has also been possible to establish the order of arrangement of 1.705 and 1.672 DNA at the heterochromaticeuchromatic junction on chromosome 3(R). A mitotic map is provided. The T_m of hybrids formed in situ showed that the hybrids were representative of the sequences being analyzed. The two satellites also were traced through a number of purification procedures to show that a covalent linkage may be likely between the 1.705 g/cc and 1.672 g/cc satellite as predicted from in situ hybridization analyses.     

Distribution of highly repeated DNA sequences in species of the genus Lens Miller.

Multiple-target fluorescence in situ hybridization (FISH) was applied on mitotic chromosomes of seven Lens taxa using two highly repetitive sequences (pLc30 and pLc7) isolated from the cultivated lentil and the multigene families for the 18S-5.8S-25S (pTa71) and 5S rRNA (pTa794) from wheat simultaneously as probes. The number and location of pLc30 and pLc7 sites on chromosomes varied markedly among the species, whereas the hybridization pattern of 5S rDNA and 18S-5.8S-25S rDNA was less variable. In general, each species showed a typical FISH karyotype and few differences were observed among accessions belonging to the same species, except for the accessions of Lens odemensis. The most similar FISH karyotype to the cultivated lentil is that of Lens culinaris subsp. orientalis, whereas Lens nigricans and Lens tomentosus are the two species that showed the most divergent FISH patterns compared with all taxa for number and location of pLc30 and 18S-5.8S-25S rDNA sites.     

Highly repeated DNA of the baboon: organization of sequences homologous to highly repeated DNA of the African green monkey.

In the African green monkey genome, 20% of the total DNA consists of a highly reiterated DNA sequence that occurs largely in long tandem arrays of a repeat unit that is 172 base-pairs in length. The DNA of the baboon contains sequences homologous to this repeat unit. However, in the baboon genome, these sequences comprise roughly 6% of the total DNA and alternate in a regular fashion with a DNA segment that may be distantly related to the monkey repeat unit. The sequences in the baboon that are homologous to the monkey repeat unit are contained within a 340 base-pair repeat unit of the highly repeated DNA fraction of the baboon. The extent of nucleotide divergence of the homologous repeated sequences between the two species is estimated to be about 10%.     

Newly evolved highly repeated DNA sequences ofTupaia glis (Tupaiidae,Scandentia)

We have studied highly repeated DNA sequences ofTupaia glis (Tupaiidae, Scandentia) with restriction endonucleases and Southern blotting techniques. Five highly repeated DNA fragments have been isolated fromT. glis and hybridized with genomic DNAs (cleaved by different restriction enzymes) of several non-human primate species and one insectivore (E. europaeus), in order to highlight eventual differences or similarities of their highly repeated DNA sequences. Our first preliminary findings suggest that the newly isolated highly repeated DNA fragments ofT. glis are distinct from both non-human primates and insectivore, the two taxonomic groups considered most similar to the Tupaiidae.     

Tobacco single-copy DNA is highly homologous to sequences present in the genomes of its diploid progenitors

Summary We compared the single-copy DNA sequences of the tetraploid tobacco plant, Nicotiana tabacum, with those of its diploid progenitors N. sylvestris and N. tomentosiformis. We observed that 65% of N. sylvestris and N. tomentosiformis single-copy DNA fragments reacted with each other using moderately stringent hybridization conditions (60° C, 0.18 M Na⁺). An additional 10% sequence homology was detected when the hybridization temperature was reduced by 10° C. The thermal stability of interspecific single-copy DNA duplexes indicated that they were approximately 6% more mispaired than homologous single-copy DNA duplexes. In contrast, we observed almost no single-copy DNA divergence between N. tabacum and its diploid progenitors. Greater than 99% of N. sylvestris and N. tomentosiformis single-copy DNAs reacted with N. tabacum DNA using moderately stringent hybridization conditions. The thermal stability of these duplexes indicated that they contained no more sequence mismatch than homologous single-copy duplexes. Together, our results show that significant single-copy DNA sequence divergence has occurred between the diploid N. sylvestris and N. tomentosiformis genomes. However, by applying our experimental criteria these single-copy DNAs are indistinguishable from their counterparts in the hybrid N. tabacum nucleus.     

Evolutionary growth process of highly conserved sequences in vertebrate genomes

Genome sequence comparison between evolutionarily distant species revealed ultraconserved elements (UCEs) among mammals under strong purifying selection. Most of them were also conserved among vertebrates. Because they tend to be located in the flanking regions of developmental genes, they would have fundamental roles in creating vertebrate body plans. However, the evolutionary origin and selection mechanism of these UCEs remain unclear. Here we report that UCEs arose in primitive vertebrates, and gradually grew in vertebrate evolution. We searched for UCEs in two teleost fishes, Tetraodon nigroviridis and Oryzias latipes, and found 554 UCEs with 100% identity over 100 bps. Comparison of teleost and mammalian UCEs revealed 43 pairs of common, jawed-vertebrate UCEs (jUCE) with high sequence identities, ranging from 83.1% to 99.2%. Ten of them retain lower similarities to the Petromyzon marinus genome, and the substitution rates of four non-exonic jUCEs were reduced after the teleost-mammal divergence, suggesting that robust conservation had been acquired in the jawed vertebrate lineage. Our results indicate that prototypical UCEs originated before the divergence of jawed and jawless vertebrates and have been frozen as perfect conserved sequences in the jawed vertebrate lineage. In addition, our comparative sequence analyses of UCEs and neighboring regions resulted in a discovery of lineage-specific conserved sequences. They were added progressively to prototypical UCEs, suggesting step-wise acquisition of novel regulatory roles. Our results indicate that conserved non-coding elements (CNEs) consist of blocks with distinct evolutionary history, each having been frozen since different evolutionary era along the vertebrate lineage.     

Highly repetitive DNA sequences in cyanobacterial genomes.

We characterized three distinct families of repeated sequences in the genome of the cyanobacterium Calothrix sp. strain PCC 7601. These repeated sequences were present at a level of about 100 copies per Calothrix genome and consisted of tandemly amplified heptanucleotides. These elements were named short tandemly repeated repetitive (STRR) sequences. We used the three different Calothrix STRR sequences as probes to perform Southern hybridization experiments with DNAs extracted from various cyanobacterial strains, Bacillus subtilis, and Escherichia coli. The three different STRR sequences were found as repetitive genomic DNA components specific to the heterocystous strains tested. The role of the STRR sequences, as well as their possible use in taxonomic studies, is discussed.     

Organization of repeated sequences in species of the genus Avena

Summary Four repetitive sequences from Avena murphyi have been isolated and their genome organization studied in different species of the genus Avena. A tandem sequence array was found for the Avena species that contain the C genome. Three other dispersed sequences present in the A and C genomes were arranged in a genomespecific manner. The fact that no major differences in the hybridization patterns were found between species with the same basic genome is consistent with the current taxonomy of Avena species.