Tnat1 and Tnat2 from Arabidopsis thaliana: novel transposable elements with tandem repeat sequences.

A computer-aided homology search of databases found that the nucleotide sequences flanking ATLN44, a non-LTR retrotransposon (LINE) from Arabidopsis thaliana, are repeated in the A. thaliana genome. These sequences are homologous to flanking sequences of 664 bp with terminal inverted repeat sequences of about 70 bp. The 664-bp sequence and most of the 14 homologues identified were flanked by direct repeat sequences of 9 bp. These findings indicate that the repeated sequence, named Tnat1, is a transposable element that duplicates a 9-bp sequence at the target site on transposition and that ATLN44 is inserted in one Tnat1 member. Interestingly, all of the Tnat1 members had tandem repeats comprised of several units of a 60-bp sequence, the number of repeats differing among Tnat1 members. Of the Tnat1 members identified, one was inserted into another sequence repeated in the A. thaliana genome: that sequence is about 770 bp long and has terminal inverted repeat sequences of about 110 bp. The sequence is flanked by direct repeats of a 9-bp sequence, indicating that it is another transposable element, named Tnat2, from A. thaliana. Moreover, Tnat2 members had a tandem repeat about 240 bp long. Tnat1 and Tnat2 with tandem repeats in their internal regions show no homology to each other or to any of the elements identified previously; therefore they appear to be novel transposable elements.     

Molecular characterization of a family of tandemly repeated DNA sequences,TR-1, in heterochromatic knobs of maize and its relatives

Two families of tandem repeats, 180-bp and TR-1, have been found in the knobs of maize. In this study, we isolated 59 clones belonging to the TR-1 family from maize and teosinte. Southern hybridization and sequence analysis revealed that members of this family are composed of three basic sequences, A (67 bp); B (184 bp) or its variants B' (184 bp), 2/3B (115 bp), 2/3B' (115 bp); and C (108 bp), which are arranged in various combinations to produce repeat units that are multiples of approximately 180 bp. The molecular structure of TR-1 elements suggests that: (1) the B component may evolve from the 180-bp knob repeat as a result of mutations during evolution; (2) B' may originate from B through lateral amplification accompanied by base-pair changes; (3) C plus A may be a single sequence that is added to B and B', probably via nonhomologous recombination; and (4) 69 bp at the 3' end of B or B', and the entire sequence of C can be removed from the elements by an unknown mechanism. Sequence comparisons showed partial homologies between TR-1 elements and two centromeric sequences (B repeats) of the supernumerary B chromosome. This result, together with the finding of other investigators that the B repeat is also fragmentarily homologous to the 180-bp repeat, suggests that the B repeat is derived from knob repeats in A chromosomes, which subsequently become structurally modified. Fluorescence in situ hybridization localized the B repeat to the B centromere and the 180-bp and TR-1 repeats to the proximal heterochromatin knob on the B chromosome.     

Comparative analysis of the nucleosomal structure of rye,wheat and their relatives

Analysis of the structure of chromatin in cereal species using micrococcal nuclease (MNase) cleavage showed nucleosomal organization and a ladder with typical nucleosomal spacing of 175–185 bp. Probing with a set of DNA probes localized in the authentic telomeres, subtelomeric regions and bulk chromatin revealed that these chromosomal regions have nucleosomal organization but differ in size of nucleosomes and rate of cleavage between both species and regions. Chromatin from Secale and Dasypyrum cleaved more quickly than that from wheat and barley, perhaps because of their higher content of repetitive sequences with hairpin structures accessible to MNase cleavage. In all species, the telomeric chromatin showed more rapid cleavage kinetics and a shorter nucleosome length (160 bp spacing) than bulk chromatin. Rye telomeric repeat arrays were shortest, ranging from 8 kb to 50 kb while those of wheat ranged from 15 kb up to 175 kb. A gradient of sensitivity to MNase was detected along rye chromosomes. The rye-specific subtelomeric sequences pSc200 and pSc250 have nucleosomes of two lengths, those of the telomeric and of bulk nucleosomes, indicating that the telomeric structure may extended into the chromosomes. More proximal sequences common to rye and wheat, the short tandem-repeat pSc119.2 and rDNA sequence pTa71, showed longer nucleosomal sizes characteristic of bulk chromatin in both species. A strictly defined spacing arrangement (phasing) of nucleosomes was demonstrated along arrays of tandem repeats with different monomer lengths (118, 350 and 550 bp) by combining MNase and restriction enzyme digestion.     

Pericentromeric regions of soybean (Glycine max L. Merr.) chromosomes consist of retroelements and tandemly repeated DNA and are structurally and evolutionarily labile

Little is known about the physical makeup of heterochromatin in the soybean (Glycine max L. Merr.) genome. Using DNA sequencing and molecular cytogenetics, an initial analysis of the repetitive fraction of the soybean genome is presented. BAC 076J21, derived from linkage group L, has sequences conserved in the pericentromeric heterochromatin of all 20 chromosomes. FISH analysis of this BAC and three subclones on pachytene chromosomes revealed relatively strict partitioning of the heterochromatic and euchromatic regions. Sequence analysis showed that this BAC consists primarily of repetitive sequences such as a 102-bp tandem repeat with sequence identity to a previously characterized approximately 120-bp repeat (STR120). Fragments of Calypso-like retroelements, a recently inserted SIRE1 element, and a SIRE1 solo LTR were present within this BAC. Some of these sequences are methylated and are not conserved outside of G. max and G. soja, a close relative of soybean, except for STR102, which hybridized to a restriction fragment from G. latifolia. These data present a picture of the repetitive fraction of the soybean genome that is highly concentrated in the pericentromeric regions, consisting of rapidly evolving tandem repeats with interspersed retroelements.     

Identification and characterization of tandem repeats in exon III of dopamine receptor D4 (DRD4) genes from different mammalian species

In this study we have identified and characterized dopamine receptor D4 (DRD4) exon III tandem repeats in 33 public available nucleotide sequences from different mammalian species. We found that the tandem repeat in canids could be described in a novel and simple way, namely, as a structure composed of 15- and 12- bp modules. Tandem repeats composed of 18-bp modules were found in sequences from the horse, zebra, onager, and donkey, Asiatic bear, polar bear, common raccoon, dolphin, harbor porpoise, and domestic cat. Several of these sequences have been analyzed previously without a tandem repeat being found. In the domestic cow and gray seal we identified tandem repeats composed of 36-bp modules, each consisting of two closely related 18-bp basic units. A tandem repeat consisting of 9-bp modules was identified in sequences from mink and ferret. In the European otter we detected an 18-bp tandem repeat, while a tandem repeat consisting of 27-bp modules was identified in a sequence from European badger. Both these tandem repeats were composed of 9-bp basic units, which were closely related with the 9-bp repeat modules identified in the mink and ferret. Tandem repeats could not be identified in sequences from rodents. All tandem repeats possessed a high GC content with a strong bias for C. On phylogenetic analysis of the tandem repeats evolutionary related species were clustered into the same groups. The degree of conservation of the tandem repeats varied significantly between species. The deduced amino acid sequences of most of the tandem repeats exhibited a high propensity for disorder. This was also the case with an amino acid sequence of the human DRD4 exon III tandem repeat, which was included in the study for comparative purposes. We identified proline-containing motifs for SH3 and WW domain binding proteins, potential phosphorylation sites, PDZ domain binding motifs, and FHA domain binding motifs in the amino acid sequences of the tandem repeats. The numbers of potential functional sites varied pronouncedly between species. Our observations provide a platform for future studies of the architecture and evolution of the DRD4 exon III tandem repeat, and they suggest that differences in the structure of this tandem repeat contribute to specialization and generation of diversity in receptor function.     

A new family of dispersed repeats from Brassica nigra: characterization and localization

The 459-bp HindIII (pBN-4) and the 1732-bp Eco RI (pBNE8) fragments from the Brassica nigra genome were cloned and shown to be members of a dispersed repeat family. Of the three major diploid Brassica species, the repeat pBN-4 was found to be highly specific for the B. nigra genome. The family also hybridized to Sinapis arvensis showing that B. nigra had a closer relationship with the S. arvensis genome than with B. oleracea or B. campestris. The clone pBNE8 showed homology to a number of tRNA species indicating that this family of repeats may have originated from a tRNA sequence. The species-specific 459-bp repeat pBN-4 was localized on the B. nigra chromosomes using monosomic addition lines. In addition to the localization of pBN-4, the chromosomal distribution of two other species-specific repeats, pBN34 and pBNBH35 (reported earlier), was studied. The dispersed repeats pBN-4 and pBNBH35 were found to be present on all of the chromosomes, whereas the tandem repeat pBN34 was localized on two chromosomes.     

Chromosomal organization of a sequence related to LTR-like elements of Ty1-copia retrotransposons in Avena species.

A repetitive sequence, pAs17, was isolated from Avena strigosa (As genome) and characterized. The insert was 646 bp in length and showed 54% AT content. Databank searches revealed its high homology to the long terminal repeat (LTR) sequences of the specific family of Ty1-copia retrotransposons represented by WIS2-1A and Bare. It was also found to be 70% identical to the LTR domain of the WIS2-1A retroelement of wheat and 67% identical to the Bare-1 retroelement of barley. Southern hybridizations of pAs17 to diploid (A or C genomes), tetraploid (AC genomes), and hexaploid (ACD genomes) oat species revealed that it was absent in the C diploid species. Slot-blot analysis suggested that both diploid and tetraploid oat species contained 1.3 x 10(4) copies, indicating that they are a component of the A-genome chromosomes. The hexaploid species contained 2.4 x 10(4) copies, indicating that they are a component of both A- and D-genome chromosomes. This was confirmed by fluorescent in situ hybridization analyses using pAs17, two ribosomal sequences, and a C-genome specific sequence as probes. Further, the chromosomes involved in three C-A and three C-D intergenomic translocations in Avena murphyi (AC genomes) and Avena sativa cv. Extra Klock (ACD genomes), respectively, were identified. Based on its physical distribution and Southern hybridization patterns, a parental retrotransposon represented by pAs17 appears to have been active at least once during the evolution of the A genome in species of the Avena genus.     

A new rice repetitive DNA shows sequence homology to both 5S RNA and tRNA.

Moderately repetitive DNA sequences are found in the genomes of all eucaryotes that have been examined. We now report the discovery of a novel, transcribed, moderately repetitive DNA sequence in a higher plant which is different from any of the known repetitive DNA sequences from any organism. We isolated a rice cDNA clone which hybridizes to multiple bands on genomic blot analysis. The sequence of this 352 bp cDNA contains four regions of homology to the wheat phenylalanine tRNA, including the polymerase III-type promoter. Unexpectedly, two regions of the same 352 bp sequence also show homology to the wheat 5S RNA sequence. Using the cDNA as a probe, we have isolated six genomic clones which contain long tandem repeats of 355 bp sequence, and have sequenced nine repeat units. Our findings suggest that the rice repetitive sequence may be an amplified pseudogene with sequence homology to both 5S RNA and tRNA, but organized as long tandem repeats resembling 5S RNA genes. This is the first example showing homology between the sequences of a moderately repetitive DNA with unknown function and 5S RNA.     

Molecular cloning and characterization of novel centromeric repetitive DNA sequences in the blue-breasted quail (Coturnix chinensis,Galliformes)

A new family of centromeric highly repetitive DNA sequences was isolated from EcoRI-digested genomic DNA of the blue-breasted quail (Coturnix chinensis, Galliformes), and characterized by filter hybridization and chromosome in situ hybridization. The repeated elements were divided into two types by nucleotide length and chromosomal distribution; the 578-bp element predominantly localized to microchromosomes and the 1,524-bp element localized to chromosomes 1 and 2. The 578-bp element represented tandem arrays and did not hybridize to genomic DNAs of other Galliformes species, chicken (Gallus gallus), Japanese quail (Coturnix japonica) and guinea fowl (Numida meleagris). On the other hand, the 1,524-bp element was not organized in tandem arrays, and did hybridize to the genomic DNAs of three other Galliformes species, suggesting that the 1,524-bp element is highly conserved in the Galliformes. The 578-bp element was composed of basic 20-bp internal repeats, and the consensus nucleotide sequence of the internal repeats had homologies to the 41-42 bp CNM repeat and the XHOI family repeat of chicken. Our data suggest that the microchromosome-specific highly repetitive sequences of the blue-breasted quail and chicken were derived from a common ancestral sequence, and that they are one of the major and essential components of chromosomal heterochromatin in Galliformes species.     

Molecular cytogenetics and tandem repeat sequence evolution in the allopolyploid Nicotiana rustica compared with diploid progenitors N. paniculata and N. undulata

Nicotiana rustica (2n = 4x = 48) is a natural allotetraploid composed of P and U genomes which are closely related to genomes of diploid species N. paniculata and N. undulata. Genomic in situ hybridization (GISH) also confirms that the diploid parents, or close relatives, are the ancestors of N. rustica. In order to study genetic interactions between ancestral genomes in the allotetraploid, we isolated three families of repetitive sequences, two from N. paniculata (NPAMBE and NPAMBO) and one from N. undulata (NUNSSP). Southern blot hybridization revealed that the sequences are digested with a range of restriction enzymes into regular ladder patterns indicating a tandem arrangement of high copy repeats possessing monomeric units of about 180 bp. The three-tandem sequences belong to a larger Nicotiana tandem repeat family called here the HRS-60 family. Members of this family are found in all Nicotiana species studied. Fluorescence in situ hybridization (FISH) analysis localized the satellite repeats to subtelomeric regions of most chromosomes of N. paniculata and N. undulata. The pattern of sequence distribution on the P- and U-genomes of N. rustica was similar to the putative parents N. paniculata and N. undulata respectively. However, NPAMBO repeats appear to be reduced and rearranged in N. rustica that may suggest evolution within the P genome. GISH and FISH with the tandem repeat probes failed to reveal intergenomic translocations as might be predicted from the nucleocytoplasmic interaction hypothesis.     

The heterochromatin of grasshoppers from the Caledia captiva species complex. I. Sequence evolution and conservation in a highly repeated DNA family

The restriction enzyme TaqI digests 0.2% of the genomic DNA from the grasshopper Caledia captiva to a family of sequences 168 bp in length (length of consensus sequence). The sequence variation of this "Taq family" of repeat units was examined among four races from C. captiva to assay the pattern of evolution within this highly repeated DNA. The Taq-family repeats are located in C-banded heterochromatin on at least one member of each homologous pair of chromosomes; the locations range from centromeric to telomeric. Thirty-nine cloned repeats isolated from two population 1A individuals along with 11 clones from seven populations taken from three of the races demonstrated sequence variation at 72 positions. Pairwise comparisons of the cloned repeats, both within an individual and between different races, indicate that levels of intraspecific divergence, as measured by reproductive incompatibility, do not correlate with sequence divergence among the 168-bp repeats. A number of subsequences within the repeat remain unchanged among all 50 clones; the longest of these is 18 bp. That the same 18-bp subsequence is present in all clones examined is a finding that departs significantly (P less than 0.01) from what would be expected to occur at random. Two other cloned repeats, from a reproductively isolated race of C. captiva, have sequences that show 56% identity with this 18-bp conserved region. An analysis showed that the frequency of occurrence of an RsaI recognition site within the 168- bp repeat in the entire Taq family agreed with that found in the cloned sequences. These data, along with a partial sequence for the entire Taq family obtained by sequencing uncloned repeats, suggest that the consensus sequence from the cloned copies is representative of this highly repeated family and is not a biased sample resulting from the cloning procedure. The 18-bp conserved sequence is part of a 42-bp sequence that possesses dyad symmetry typical of protein-binding sites. We speculate that this may be significant in the evolution of the Taq family of sequences.      

Structural heterogeneity in the R173 family of rye-specific repetitive DNA sequences

The rye-specific R173 family of repeated DNA sequences consists of ca. 15 000 individual copies per diploid rye (Secale cereale) genome and is distributed over all 7 rye chromosomes in a dispersed manner. Individual R173 elements vary in size between 3 and 6 kb, are generally not arranged as tandem repeats and are flanked by both multi-copy and single-copy sequences. DNA sequence analysis of three R173 elements (R173-1, R173-2 and R173-3) demonstrated a high degree of homology in conserved domains. The structure of R173-1 was quite different from the other two elements: long direct repeats, which represent a rye-specific repetitive sequence, were found at the ends and a 600 bp long domain was replaced by an unrelated sequence of approximately equal size. R173-2 and R173-3 were extremely similar to each other with the exception of a terminal truncation of R173-2. No open reading frames for proteins >20 kDa were present and a database search failed to detect significant homologies to published protein sequences. Despite the transposon like genomic organisation of the R173 family, individual elements lacked sequence features frequently associated with transposons and retrotransposons. In contrast, two of the regions flanking R173 elements showed strong DNA homologies to a 850 bp long region of a proposed wheat retrotransposon and to a 300 bp long region downstream of the wheatGlu-D1 gene.     

Genomic organization of human 5 S rDNA and sequence of one tandem repeat

An organization of human 5 S rDNA repeats is inferred from Southern analyses of restriction digests of genomic DNA fractionated by pulsed-field and conventional gel electrophoreses. A single unit of 2.2 kb is repeated approximately 90 times within a 200-kb fragment (defined by enzymes that do not cleave within individual units, i.e., EcoR1, BglII, HindIII, and PvuII); a comparable number of 5 S sequences are scattered elsewhere in the genome. A lambda clone containing six complete 5 S repeats was obtained from a human placental DNA library. One repeat contains 2231 bp and includes poly(dG-dT).(dC-dA), tracts of polypyrimidine, and an Alu sequence in the spacer region. Also, 5-S-hybridizing clones, containing DNA inserts with an average size of 250 kb, have been obtained as yeast artificial chromosomes. Thus far, four clones have been partially characterized and shown to be 5 S sequences from loci separate from the tandem repeat units.     

A new gypsy-type retrotransposon, RIRE7: preferential insertion into the tandem repeat sequence TrsD in pericentromeric heterochromatin regions of rice chromosomes

A portion of an insertion sequence present in a member of the RIRE3 family of retrotransposons in Oryza sativa L. cv. IR36 was found to have an LTR sequence followed by a PBS sequence complementary to the 3'-end region of tRNAMet, indicative of another rice retrotransposon (named RIRE7). Cloning and sequencing of PCR-amplified fragments that made up all parts of the RIRE7 sequence showed that RIRE7 is a gypsy-type retrotransposon with partial homology in the pol region to the rice gypsy-type retrotransposons RIRE2 and RIRE3 identified in rice previously. Interestingly, various portions of the RIRE7 sequence were homologous to several DNA segments present in the centromere regions of cereal chromosomes. Further cloning and nucleotide sequencing of fragments flanking RIRE7 copies showed that RIRE7 was inserted into a site within a tandem repeat sequence that has a unit length of 155 bp. The tandem repeat sequence, named TrsD, was homologous to tandem repeat sequences RCS2 and CentC, previously identified in the centromeric regions of rice and maize chromosomes. Fluorescence in situ hybridization (FISH) analysis of the metaphase chromosomes of O. sativa cv. Nipponbare showed that both RIRE7 and TrsD sequences were present in the centromere regions of the chromosomes. The presence of RIRE7 and the TrsD sequences in the centromere regions of several chromosomes was confirmed by the identification of several YAC clones whose chromosomal locations are known. Further FISH analysis of rice pachytene chromosomes showed that the TrsD sequences were located in a pericentromeric heterochromatin region. These findings strongly suggest that RIRE7 and TrsD are components of the pericentromeric heterochromatin of rice chromosomes.     

St基因组中的CRW同源序列在偃麦草中的FISH分析

为了确定十倍体长穗偃麦草(Thinopyrum ponticum, Liu & Wang)和六倍体中间偃麦草(Th. intermedium, [Host] Barkworth & Dewey )的基因组组成, 根据野生一粒小麦(Triticum boeoticum)着丝粒自主型反转录转座子(CRW)序列设计特异引物, 以二倍体拟鹅观草(Pseudoroegneria spicata, Á Löve )基因组 DNA为模板进行PCR扩增, 筛选到一条St基因组着丝粒区相对特异反转录转座子的部分序列pStC1, 长度为1.755 kb (GenBank登录号: FJ952565), 其中有800 bp与小麦着丝粒反转录转座子(CRW)的LTR区高度同源, 另有小部分片段与其外壳蛋白编码基因(gag)部分同源, 并且包含一段富含AGCAAC碱基的重复序列。以pStC1为探针, 对十倍体长穗偃麦草的FISH检测结果显示其基因组组成为两个St组3个E组(St₁St₂E^eE^bE^x); pStC1与中间偃麦草杂交时, 不仅St基因组上有强烈的荧光信号, 而且E基因组一些染色体的近着丝粒区域也有杂交信号, 说明偃麦草属异源多倍体物种在其形成及进化过程中St与E基因组之间在着丝粒及近着丝粒相关区域可能存在协同进化。     

Comparative analysis of tandem repeats from hundreds of species reveals unique insights into centromere evolution

Background

Centromeres are essential for chromosome segregation, yet their DNA sequences evolve rapidly. In most animals and plants that have been studied, centromeres contain megabase-scale arrays of tandem repeats. Despite their importance, very little is known about the degree to which centromere tandem repeats share common properties between different species across different phyla. We used bioinformatic methods to identify high-copy tandem repeats from 282 species using publicly available genomic sequence and our own data.

Results

Our methods are compatible with all current sequencing technologies. Long Pacific Biosciences sequence reads allowed us to find tandem repeat monomers up to 1,419 bp. We assumed that the most abundant tandem repeat is the centromere DNA, which was true for most species whose centromeres have been previously characterized, suggesting this is a general property of genomes. High-copy centromere tandem repeats were found in almost all animal and plant genomes, but repeat monomers were highly variable in sequence composition and length. Furthermore, phylogenetic analysis of sequence homology showed little evidence of sequence conservation beyond approximately 50 million years of divergence. We find that despite an overall lack of sequence conservation, centromere tandem repeats from diverse species showed similar modes of evolution.

Conclusions

While centromere position in most eukaryotes is epigenetically determined, our results indicate that tandem repeats are highly prevalent at centromeres of both animal and plant genomes. This suggests a functional role for such repeats, perhaps in promoting concerted evolution of centromere DNA across chromosomes.