Characterization and chromosomal organization of satellite DNA sequences in Picea abies

Three clones containing satellite DNA sequences were selected from a randomly sheared genomic DNA library of Picea abies (clones PAF1, PAG004P22F (2F), and PAG004E03C (3C)). PAF1 contained 7 repeats that were 37-55 bp in length and had 68.9%-91.9% nucleotide sequence similarity. Two 2F repeats were 305-306 bp in length and had 83% sequence similarity. Two 3C repeats were 193-226 bp in length and had a sequence similarity of 78.6%. The copy number per 1C DNA of PAF1, 2F, and 3C repeats was 2.7 x 106, 2.9 x 105, and 2.9 x 104, respectively. In situ hybridization showed centromeric localization of these sequences in two chromosome pairs with PAF1, all pairs but one with 2F, and three pairs with 3C. Moreover, PAF1 sequences hybridized at secondary constrictions in six pairs, while 2F-related sequences were found at these chromosome regions only in four pairs. These hybridization patterns allow all chromosome pairs to be distinguished. PAF1-related repeats were contained in the intergenic spacer (IGS) of ribosomal cistrons in all six nucleolar organizers of the complement, while sequences related to 2F were found on only one side of the rDNA arrays in four pairs, showing structural diversity between rDNA regions of different chromosomes.     

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.     

A comparative study of genomes in angiosperms

Angiosperms investigated by DNA/DNA reassociation studies were classified and tested for a taxonomic class- and subclass-specifity in a biometrical fashion. Monocotyledons and Dicotyledons differ significantly from each other with respect to a genomic parameter (U/R-ratio;U single copy DNA fraction;R = 1-U fraction of repetitive DNA). This difference is discussed from an evolutionary and molecular point of view.—Intercorrelations between the fraction of fast repeats, slow repeats, and single copy DNA can be detected. The amount of DNA organized in a short period pattern of interspersion is found to depend on the fraction of repetitive and single copy DNA. The number of DNA segments tandemly arranged in a short period pattern is linearly correlated withR/U-values. This correlation allows for a formula suitable for the estimation of the number of active genes in angiosperms. The analytical complexities of repetitive and single copy DNA are linearly correlated with the genome size of higher plants. The ratioU/R depends on the genome size of angiosperms in a hyperbolic fashion.     

Genomic organization and evolution of the soybean SB92 satellite sequence

Repetitive DNA sequences comprise a large percentage of plant genomes, and their characterization provides information about both species and genome evolution. We have isolated a recombinant clone containing a highly repeated DNA element (SB92) that is homologous to ca. 0.9% of the soybean genome or about 10⁵ copies. This repeated sequence is tandemly arranged and is found in four or five major genomic locations. FISH analysis of metaphase chromosomes suggests that two of these locations are centromeric. We have determined the sequence of two cloned repeats and performed genomic sequencing to obtain a consensus sequence. The consensus repeat size was 92 bp and exhibited an average of 10% nucleotide substitution relative to the two cloned repeats. This high level of sequence diversity suggests an ancient origin but is inconsistent with the limited phylogenetic distribution of SB92, which is found an high copy number only in the annual soybeans. It therefore seems likely that this sequence is undergoing very rapid evolution.     

Highly reiterated non-coding sequence in the genome of Plasmodium falciparum is composed of 21 base-pair tandem repeats

Clones containing highly reiterated DNA sequences were isolated from a Plasmodium falciparum genomic library. One clone, Rep2, was selected for further characterization by nucleotide sequence analysis. The results revealed that the insert of this clone is composed of tandemly arranged 21 base-pair imperfect repeats. These repeats are estimated to comprise approximately 1% of the P. falciparum genome and there are 10(4) to 2 X 10(5) copies, depending on the genome size estimate used for calculation. Moreover, the repeats are organized in clusters and do not appear to be transcribed in non-synchronized P. falciparum cultures.     

The repair of double-strand breaks in plants: mechanisms and consequences for genome evolution

The efficient repair of double-strand breaks (DSBs) in genomic DNA is important for the survival of all organisms. In recent years, basic mechanisms of DSB repair in somatic plant cells have been elucidated. DSBs are mainly repaired by non-homologous end-joining (NHEJ). The repair can be associated with deletions, but also insertions due to copying genomic sequences from elsewhere into the break. Species-specific differences of NHEJ have been reported and an inverse correlation of deletion size to genome size has been postulated, indicating that NHEJ might contribute significantly to evolution of genome size. DSB repair by homologous recombination (HR) might also influence genome organization. Whereas homology present in an allelic or an ectopic position is hardly used for repair, the use of homologous sequences in close proximity to the break is frequent. A 'single-strand annealing' mechanism that leads to sequence deletions between direct repeats is particularly efficient. This might explain the accumulation of single long terminal repeats of retroelements in cereal genomes. The conservative 'synthesis-dependent strand annealing' mechanism, resulting in conversions without crossovers is also prominent and seems to be significant for the evolution of tandemly arranged gene families such as resistance genes. Induction of DSBs could be used as a means for the controlled manipulation of plant genomes in an analogous way for the use of marker gene excision and site-specific integration.     

Physical arrangement of retrotransposon-related repeats in centromeric regions of wheat

Cereal centromeres commonly contain many repetitive sequences that are derived from Ty3/gypsy retrotransposon. FISH analysis using a large DNA insert library of wheat identified a 67-kb clone (R11H) that showed strong hybridization signals on the centromeres. The R11H clone contains Ty3/gypsy retrotransposon-related sequences; both integrase and CCS1 family sequences were identified. Subsequently, we isolated additional 23 large-insert clones which also contained the integrase and CCS1 sequences. Based on the number of the integrase repeats in the clones determined by DNA gel blot analysis, we concluded that the retrotransposon-like sequences are tandemly repeated in wheat centromeres in ca. 55-kb interval on average. This conclusion is consistent with the results of FISH analysis on the extended DNA fibers.     

Repetitive sequences of the sea urchin genome: Distribution of members of specific repetitive families

Three repetitive sequence families from the sea urchin genome were studied, each defined by homology with a specific cloned probe one to a few hundred nucleotides long. Recombinant λ-sea urchin DNA libraries were screened with these probes, and individual recombinants were selected that include genomic members of these families. Restriction mapping, gel blot, and kinetic analyses were carried out to determine the organization of each repeat family. Sequence elements belonging to the first of the three repeat families were found to be embedded in longer repeat sequences. These repeat sequences frequently occur in small clusters. Members of the second repeat family are also found in a long repetitive sequence environment, but these repeats usually occur singly in any given region of the DNA. The sequences of the third repeat are only 200 to 300 nucleotides long, and are generally terminated by single copy DNA, though a few examples were found associated with other repeats. These three repeat sequence families constitute sets of homologous sequence elements that relate distant regions of the DNA.     

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.     

Repetitive DNA, molecular cytogenetics and genome organization in the King scallop (Pecten maximus)

We studied the structure, organization and relationship of repetitive DNA sequences in the genome of the scallop, Pecten maximus, a bivalve that is important both commercially and in marine ecology. Recombinant DNA libraries were constructed after partial digestion of genomic DNA from scallop with PstI and ApaI restriction enzymes. Clones containing repetitive DNA were selected by hybridisation to labelled DNA from scallop, oyster and mussel; colonies showing strong hybridisation only to scallop were selected for analysis and sequencing. Six non-homologous tandemly repeated sequences were identified in the sequences, and Southern hybridisation with all repeat families to genomic DNA digests showed characteristic ladders of hybridised bands. Three families had monomer lengths around 40 bp while three had repeats characteristic of the length wrapping around one (170 bp), or two (326 bp) nucleosomes. In situ hybridisation to interphase nuclei showed each family had characteristic numbers of clusters indicating contrasting arrangements. Two of the repeats had unusual repetitions of bases within their sequence, which may relate to the nature of microsatellites reported in bivalves. The study of these rapidly evolving sequences is valuable to understand an important source of genomic diversity, has the potential to provide useful markers for population studies and gives a route to identify mechanisms of DNA sequence evolution.     

Molecular characterization and chromosomal distribution of species-specific repetitive DNA sequences from Beta corolliflora, a wild relative of sugar beet.

Repetitive DNA sequences have been isolated from a Sau3AI plasmid library of tetraploid Beta corolliflora (2n = 4x = 36), a wild relative of sugar beet (B. vulgaris). The library was screened by differential hybridization with genomic DNA of B. corolliflora and B. vulgaris. When used as probes for Southern hybridization of genomic DNA, six clones were determined to represent highly repetitive DNA families present only in the B. corolliflora genome. Five other sequences were highly repetitive in B. corolliflora and low or single copy in B. vulgaris. The insert size varied between 43 bp and 448 bp. Two sequences pBC1279 and pBC1944 displayed strong homology to a previously cloned satellite DNA from B. nana. With one exception, sequences are tandemly arranged as revealed by a typical ladder pattern after genomic Southern hybridization. The chromosomal distribution of five probes was determined by fluorescence in situ hybridization (FISH) of mitotic metaphases from B. corolliflora and a triploid hybrid between B. vulgaris and B. corolliflora. Three sequences were spread along all chromosome arms of B. corolliflora while one sequence was present on only six chromosomes. The chromosome-specific sequence pBC216 was found in close vicinity to the 5S rDNA located on B. corolliflora chromosome IV. This set of species-specific sequences has the potential to be used as probes for the identification of monosomic alien addition lines and for marker-assisted gene transfer from wild beet to cultivated beet.     

Evolutionary history of 4.5SH RNA

4.5SH RNA is a 94-nt small RNA with unknown function. This RNA is known to be present in the mouse, rat, and hamster cells; however, it is not found in human, rabbit, and chicken. In the mouse genome, the 4.5SH RNA gene is a part of a long (4.2 kb) tandem repeat ( approximately 800 copies) unit. Here, we found that 4.5SH RNA genes are present only in rodents of six families that comprise the Myodonta clade: Muridae, Cricetidae, Spalacidae, Rhizomyidae, Zapodidae, and Dipodidae. The analysis of complementary DNA derived from the rodents of these families showed general evolutionary conservation of 4.5SH RNA and some intraspecific heterogeneity of these RNA molecules. 4.5SH RNA genes in the Norway rat, mole rat, hamster and jerboa genomes are included in the repeated sequences. In the jerboa genome these repeats are 4.0-kb long and arranged tandemly, similar to the corresponding arrangements in the mouse and rat genomic DNA. Sequencing of the rat and jerboa DNA repeats containing 4.5SH RNA genes showed fast evolution of the gene-flanking sequences. The repeat sequences of the distantly related rodents (mouse and rat vs. jerboa) have no apparent similarity except for the 4.5SH RNA gene itself. Conservation of the 4.5SH RNA gene nucleotide sequence indicates that this RNA is likely to be under selection pressure and, thus, may have a function. The repeats from the different rodents have similar lengths and contain many simple short repeats. The data obtained suggest that long insertions, deletions, and simple sequence amplifications significantly contribute in the evolution of the repeats containing 4.5SH RNA genes. The 4.5SH RNA gene seems to have originated 50-85 MYA in a Myodonta ancestor from a copy of the B1 short interspersed element. The amplification of the gene with the flanking sequences could result from the supposed cellular requirement of the intensive synthesis of 4.5SH RNA. Further Myodonta evolution led to dramatic changes of the repeat sequences in every lineage with the conservation of the 4.5SH RNA genes only. This gene, like some other relatively recently originated genes, could be a useful model for studying generation and evolution of non-protein-coding genes.     

Effective Isolation of Retrotransposons and Repetitive DNA Families from the Wheat Genome

New classes of repetitive DNA elements were effectively identified by isolating small fragments of the elements from the wheat genome. A wheat A genome library was constructed from Triticum monococcum by degenerate cleavage with EcoO109I, the recognition sites of which consisted of 5'-PuGGNCCPy-3'multi-sequences. Three novel repetitive sequences pTm6, pTm69 and pTm58 derived from the A genome were screened and tested for high copy number using a blotting approach. pTm6 showed identity with integrase domains of the barley Ty1-Copia-retrotransposon BARE-1 and pTm58 showed similarity to the barley Ty3-gypsy-like retrotransposon Romani. pTm69, however, constituted a tandem array with useful genomic specificities, but did not share any identity with known repetitive elements. This study also sought to isolate wheat D-genome-specific repetitive elements regardless of the level of methylation, by genomic subtraction. Total genomic DNA of Aegilops tauschii was cleaved into short fragments with a methylation-insensitive 4 bp cutter, Mbol, and then common DNA sequences between Ae. tauschii and Triticum turgidum were subtracted by annealing with excess T. turgidum genomic DNA. The D genome repetitive sequence pAt1 was isolated and used to identify an additional novel repetitive sequence family from wheat bacterial artificial chromosomes with a size range of 1 395-1 850 bp. The methods successfully led pathfinding of two unique repetitive families.     

Characterization of the chromosome complement of Helianthus annuus by in situ hybridization of a tandemly repeated DNA sequence.

A tandemly repeated sequence isolated from a clone (HAG004N15) of a nebulized genomic DNA library of sunflower (Helianthus annuus L., 2n = 34) was characterized and used to study the chromosome complement of sunflower. HAG004N15 repeat units (368 bp in length) were found to be highly methylated, and their copy number per haploid (1C) genome was estimated to be 7800. After in situ hybridization of HAG004N15 repeats onto chromosome spreads, signals were observed at the end of both chromosome arms in 4 pairs and at the end of only one arm in 8 other pairs. Signals were also observed at the intercalary (mostly subtelomeric) regions in all pairs, in both arms in 8 pairs, and in only one arm in the other 9 pairs. The short arm of 1 pair was labelled entirely. The chromosomal location of ribosomal DNA was also studied by hybridizing the wheat ribosomal probe pTa71. Four chromosome pairs contained ribosomal cistrons at the end of their shorter arm, but a satellite was seen in only 3 pairs. These hybridization patterns were the same in the 3 sunflower lines studied (HA89, RA20031, and HOR). The chromosomal localization of HAG004N15-related sequences allowed all of the chromosome pairs to be distinguished from each other, in spite of small size and similar morphology.     

Genomic organization, evolution, and structural peculiarities of highly repetitive DNA of Hordeum vulgare

A fraction of highly repeated DNA sequences of Hordeum vulgare has been investigated by cloning 19 separate highly repetitive sequences in the plasmid pBR327. Characteristics studied included genus specificity of isolated sequences, their prevalence, and genome organization. Sequences (pHv7161, pHv7191, pHv7179) have been identified that are the most widespread in the H. vulgare genome and have a complicated arrangement. A tandemly arranged sequence, pHv7141, was also identified. The primary structure of a 999 bp long, BamHI fragment of one of the most widespread sequences, pHv7161, as well as the adjacent pHv7302 and pHv7245 sequences was determined. The fragment abounds in inverted repeats, of which two are flanked by direct repeats, and contains short subrepeats, A, B, and C, and a great variety of potential protein-binding sites. A comparison is drawn between the content and genome organization of highly repeated DNA sequences of H. vulgare and those of the wild barley species Hordeum bulbosum, Hordeum jubatum, Hordeum geniculatum, Hordeum brevisubulatum, Hordeum turkestanicum, and Hordeum murinum. According to the above characters (close copy number and genome organization similarity of highly repetitive sequences) the species under discussion have been classified into four groups. This division is in good agreement with other data on interspecific crossing in Hordeum and on chromosome pairing in hybrid meiosis.     

Tempo and mode of Ty element evolution in Saccharomyces cerevisiae

The Saccharomyces cerevisiae genome contains five families of long terminal repeat (LTR) retrotransposons, Ty1-Ty5. The sequencing of the S. cerevisiae genome provides an unprecedented opportunity to examine the patterns of molecular variation existing among the entire genomic complement of Ty retrotransposons. We report the results of an analysis of the nucleotide and amino acid sequence variation within and between the five Ty element families of the S. cerevisiae genome. Our results indicate that individual Ty element families tend to be highly homogenous in both sequence and size variation. Comparisons of within-element 5' and 3' LTR sequences indicate that the vast majority of Ty elements have recently transposed. Furthermore, intrafamily Ty sequence comparisons reveal the action of negative selection on Ty element coding sequences. These results taken together suggest that there is a high level of genomic turnover of S. cerevisiae Ty elements, which is presumably in response to selective pressure to escape host-mediated repression and elimination mechanisms.     

Diploidization and genome size change in allopolyploids is associated with differential dynamics of low‐ and high‐copy sequences

Recent advances have highlighted the ubiquity of whole‐genome duplication (polyploidy) in angiosperms, although subsequent genome size change and diploidization (returning to a diploid‐like condition) are poorly understood. An excellent system to assess these processes is provided by Nicotiana section Repandae, which arose via allopolyploidy (approximately 5 million years ago) involving relatives of Nicotiana sylvestris and Nicotiana obtusifolia. Subsequent speciation in Repandae has resulted in allotetraploids with divergent genome sizes, including Nicotiana repanda and Nicotiana nudicaulis studied here, which have an estimated 23.6% genome expansion and 19.2% genome contraction from the early polyploid, respectively. Graph‐based clustering of next‐generation sequence data enabled assessment of the global genome composition of these allotetraploids and their diploid progenitors. Unexpectedly, in both allotetraploids, over 85% of sequence clusters (repetitive DNA families) had a lower abundance than predicted from their diploid relatives; a trend seen particularly in low‐copy repeats. The loss of high‐copy sequences predominantly accounts for the genome downsizing in N. nudicaulis. In contrast, N. repanda shows expansion of clusters already inherited in high copy number (mostly chromovirus‐like Ty3/Gypsy retroelements and some low‐complexity sequences), leading to much of the genome upsizing predicted. We suggest that the differential dynamics of low‐ and high‐copy sequences reveal two genomic processes that occur subsequent to allopolyploidy. The loss of low‐copy sequences, common to both allopolyploids, may reflect genome diploidization, a process that also involves loss of duplicate copies of genes and upstream regulators. In contrast, genome size divergence between allopolyploids is manifested through differential accumulation and/or deletion of high‐copy‐number sequences.     

A subtelomeric satellite DNA family isolated from the genome of the dioecious plant Silene latifolia.

In an ongoing effort to trace the evolution of the sex chromosomes of Silene latifolia, we have searched for the existence of repetitive sequences specific to these chromosomes in the genome of this species by direct isolation from low-melting agarose gels of satellite DNA bands generated by digestion with restriction enzymes. Five monomeric units belonging to a highly repetitive family isolated from Silene latifolia, the SacI family, have been cloned and characterized. The consensus sequence of the repetitive units is 313 bp in length (however, high variability exists for monomer length variants) and 52.9% in AT. Repeating units are tandemly arranged at the subtelomeric regions of the chromosomes in this species. The sequence does not possess direct or inverted sequences of significant length, but short direct repeats are scattered throughout the monomer sequence. Several short sequence motives resemble degenerate monomers of the telomere repeat sequence of plants (TTTAGGG), confirming a tight association between this subtelomeric satellite DNA and the telomere repeats. Our approach in this work confirms that SacI satellite DNA sequences are among the most abundant in the genome of S. latifolia and, on the other hand, that satellite DNA sequences specific of sex chromosomes are absent in this species. This agrees with a sex determination system less cytogenetically diverged from a bisexual state than the system present in other plant species, such as R. acetosa, or at least a lesser degree of differentiation between the sex chromosomes of S. latifolia and the autosomes.