Tandem Repeat Family Research Articles

Length of uninterrupted repeats determines instability at the unstable mouse expanded simple tandem repeat family MMS10 derived from independent SINE B1 elements.

Mouse expanded simple tandem repeats (ESTRs) provide highly informative loci for analyzing spontaneous and induced germline mutation. We have conducted an extensive sequence database search and identified 17 new members of the highly unstable rodent-specific ESTR family called MMS10. This family has arisen by independent expansions of a common GGCAGA repeat unit from within a subset of both ancestral and modern SINE B1 elements during the course of mouse evolution. Analysis of the interspersion patterns of variant repeats along alleles of 20 of these MMS10 loci revealed two distinct classes of tandem arrays: one composed of uninterrupted GGCAGA repeats and the second with generally larger arrays interrupted by variant units. Surveys of allelic diversity at 11 representative members of these two classes of loci in various laboratory strains and BXD recombinant inbred lines revealed that the level of repeat instability was positively correlated with the length of uninterrupted repeats. Turnover processes at MMS10 loci, therefore, appear similar to the type of mechanism observed at human microsatellites. The MMS10 family thus provides a potentially useful murine model for studying dynamic mutation at simple tandem repeats.

Distribution and sequence analysis of the centromere‐associated repetitive element CEN38 of Sorghum bicolor (Poaceae)

Fluorescence in situ hybridization (FISH) of a large-insert genomic clone, BAC 22B2, previously suggested that Sorghum bicolor (2n = 20) has the tetraploid architecture A(b)A(b)B(b)B(b). Here, we report on BAC 22B2 subclone pCEN38 (1047-bp insert) as related to sorghum and sugarcane. Mitotic FISH of six different subclones of BAC 22B2 showed that pCEN38 produced the strongest specificity to the A(b) subgenome and signal occurred primarily near centromeres. Southern blots of pCEN38 to 21 crop plants revealed a narrow taxonomic distribution. Meiotic metaphase I FISH positioned pCEN38 sequences near active centromeres. Pachytene FISH revealed that the distributions are trimodal in several B(b) and possibly all sorghum chromosomes. DNA sequencing revealed that the pCEN38 fragment contains three tandemly repeated dimers (<280 bp) of the same sequence family found in sorghum clone pSau3A10, and that each dimer consists of two divergent monomers (<140 bp). Sequence comparisons revealed homology between the pCEN38 monomers and the SCEN 140 bp tandem repeat family of sugarcane. FISH of pCEN38 yielded signal in centromere regions of most but not all sugarcane chromosomes. Results suggest that sugarcane and sorghum share at least one ancestor harboring elements similar to pCEN38 and SCEN and that each species had an ancestor in which the repetitive element was weakly present or lacking.

Molecular characterization and distribution of a 145-bp tandem repeat family in the genus Populus.

This report aims to describe the identification and molecular characterization of a 145-bp tandem repeat family that accounts for nearly 1.5% of the Populus genome. Three members of this repeat family were cloned and sequenced from Populus deltoides and P. ciliata. The dimers of the repeat were sequenced in order to confirm the head-to-tail organization of the repeat. Hybridization-based analysis using the 145-bp tandem repeat as a probe on genomic DNA gave rise to ladder patterns which were identified to be a result of methylation and (or) sequence heterogeneity. Analysis of the methylation pattern of the repeat family using methylation-sensitive isoschizomers revealed variable methylation of the C residues and lack of methylation of the A residues. Sequence comparisons between the monomers revealed a high degree of sequence divergence that ranged between 6% and 11% in P. deltoides and between 4.2% and 8.3% in P. ciliata. This indicated the presence of sub-families within the 145-bp tandem family of repeats. Divergence was mainly due to the accumulation of point mutations and was concentrated in the central region of the repeat. The 145-bp tandem repeat family did not show significant homology to known tandem repeats from plants. A short stretch of 36 bp was found to show homology of 66.7% to a centromeric repeat from Chironomus plumosus. Dot-blot analysis and Southern hybridization data revealed the presence of the repeat family in 13 of the 14 Populus species examined. The absence of the 145-bp repeat from P. euphratica suggested that this species is relatively distant from other members of the genus, which correlates with taxonomic classifications. The widespread occurrence of the tandem family in the genus indicated that this family may be of ancient origin.

Amount and organization of the heterochromatin in Olea europaea and related species.

The amount and spatial organization of the heterochromatin in nuclei of the shoot meristem and the frequency in the nuclear DNA of sequences belonging to a family of tandem repeats were investigated in cultivars of Olea europaea and related species. Significant differences between Olea species and between cultivars of O. europaea were observed: (i) in the spatial organization of the heterochromatin in interphase nuclei as determined by the number and surface area of the chromocentres; (ii) in genome size; and (iii) in the amount of condensed chromatin as measured by cytophotometry carried out at different thresholds of optical density. DNA elements belonging to a family of tandem repeats about 80 bp in length (OeTaq80 repeats) were isolated from the genomic DNA of an olive cultivar. It was shown: (i) by nucleotide sequence comparisons, that these repeats display variability in structure even within the same array, where different elements may share no more than 74% homology; (ii) by in situ hybridization, that OeTaq80-related DNA sequences are mainly localized in the heterochromatin at the chromosome ends; (iii) by dot-blot hybridization experiments, that these sequences are highly represented in the genome of all the olive cultivars and the majority of Olea species studied, and that their frequency may differ significantly even between olive cultivars; and (iv) by calculating the copy number of OeTaq80-related sequences per haploid (1C) genome, that the redundancy of these DNA elements may differ significantly between the genomes tested. It is suggested that the inter- and intraspecific changes in the nuclear and genomic traits observed can contribute to the understanding of the phylogenetic relationships between Olea species and in defining parameters to be exploited in varietal identification within cultivated olives.

Structural analysis and physical mapping of a pericentromeric region of chromosome 5 of Arabidopsis thaliana.

The Arabidopsis thaliana CIC YAC 2D2, 510 kb long and containing a small block of 180 bp satellite units was subcloned after EcoR1 digestion in the pBluescript plasmid. One of these clones was mapped genetically in the pericentromeric region of chromosome 5. The analysis of 40 subclones of this YAC showed that they all contain repeated sequences with a high proportion of transposable elements. Three new retrotransposons, two Ty-3 Gypsy-like and one Ty-1 Copia, were identified in addition to two new tandem-repeat families. A physical map of the chromosome 5 pericentromeric region was established using CIC YAC clones, spanning around 1000 kb. This contig extends from the CIC YAC 9F5 and 7A2 positioned on the left arm of chromosome 5 to a 5S rDNA genes block localized by in-situ hybridization in the pericentromeric region. Hybridization of the subclones on the CIC YAC library showed that some of them are restricted to the pericentromeric region of chromosome 5 and represent specific markers of this region.

Molecular characterization and distribution of a 145-bp tandem repeat family in the genus &lt;i&gt;Populus&lt;/i&gt;

Molecular characterization and distribution of a 145-bp tandem repeat family in the genus &lt;i&gt;Populus&lt;/i&gt;

Chromosome-specific molecular organization of maize (Zea mays L.) centromeric regions.

A set of oat-maize chromosome addition lines with individual maize (Zea mays L.) chromosomes present in plants with a complete oat (Avena sativa L.) chromosome complement provides a unique opportunity to analyze the organization of centromeric regions of each maize chromosome. A DNA sequence, MCS1a, described previously as a maize centromere-associated sequence, was used as a probe to isolate cosmid clones from a genomic library made of DNA purified from a maize chromosome 9 addition line. Analysis of six cosmid clones containing centromeric DNA segments revealed a complex organization. The MCS1a sequence was found to comprise a portion of the long terminal repeats of a retrotransposon-like repeated element, termed CentA. Two of the six cosmid clones contained regions composed of a newly identified family of tandem repeats, termed CentC. Copies of CentA and tandem arrays of CentC are interspersed with other repetitive elements, including the previously identified maize retroelements Huck and Prem2. Fluorescence in situ hybridization revealed that CentC and CentA elements are limited to the centromeric region of each maize chromosome. The retroelements Huck and Prem2 are dispersed along all maize chromosomes, although Huck elements are present in an increased concentration around centromeric regions. Significant variation in the size of the blocks of CentC and in the copy number of CentA elements, as well as restriction fragment length variations were detected within the centromeric region of each maize chromosome studied. The different proportions and arrangements of these elements and likely others provide each centromeric region with a unique overall structure.

Evolutionarily conserved telomeric location of BBC1 and MC1R on a microchromosome questions the identity of MC1R and a pigmentation locus on chromosome 1 in chicken.

per haploid genome (Gazdova et al. 1995). The NTRS family comprises monomeric units of 212±219 bp and is present in approximately 8 3 10 copies per haploid genome (MatyaÂsek et al. 1997). The GRS family is present in intercalary sites of ®ve chromosome pairs of the T subgenome (Gazdova et al. 1995). The NTRS sequence is speci®c for the T1 chromosome, where it occupies an intercalary site (MatyaÂek et al. 1997). In summary, the NTS9 family is distinguished from the three other tabacco tandem repeat families by its sequence, shorter unit length and chromosomal location. To our knowledge, the NTS9 sequence is the ®rst tandemly repeated sequence of tobacco to be localized close to a centromere and to be highly enriched on the S9 chromosome of the S subgenome. Acknowledgements

Macromolecular organization and genetic mapping of a rapidly evolving chromosome-specific tandem repeat family (B77) in cotton (Gossypium).

Isolation and characterization of the most prominent repetitive element families in the genome of tetraploid cotton (Gossypium barbadense L; [39]) revealed a small subset of families that showed very different properties in tetraploids than in their diploid progenitors, separated by 1-2 million years. One element, B77, was characterized in detail, and compared to the well-conserved 5S and 45S rRNA genes. The 572 bp B77 repeat was found to be concentrated in several discontinuous tandem arrays confined to a single 550 kb SalI fragment in tetraploid cotton. Genetic mapping based on the absence of the pentameric 'rung' in the G. barbadense 'ladder' showed that B77 maps to a D-subgenome chromosome. In situ hybridization supports the contention that the array is confined largely to a single chromosomal site in the D-subgenome. The B77 repeat has undergone a substantial increase in copy number since formation of tetraploid cotton from its diploid relatives. RFLPs observed among tetraploid cotton species suggest that amplification and/or rearrangement of the repeat may have continued after divergence of the five tetraploid cotton species. B77 contains many short direct repeats and shares significant DNA sequence homology with a Nicotiana alata retrotransposon Tna1-2 integrase motif. The recent amplification of B77 on linkage group D04 suggests that the D-subgenome of tetraploid cotton may be subject to different evolutionary constraints than the D-genome diploid chromosomes, which exhibit few genome-specific elements. Further, the abundance of B77 in G. gossypioides supports independent evidence that it may be the closest extant relative of the D-genome ancestor of cotton.

Correction: BEHAB, a new member of the proteoglycan tandem repeat family of hyaluronan-binding proteins that is restricted to the brain

Correction: BEHAB, a new member of the proteoglycan tandem repeat family of hyaluronan-binding proteins that is restricted to the brain

A novel species-specific tandem repeat DNA family from Sinapis arvensis: detection of telomere-like sequences.

DNA sequences representing a tandemly repeated DNA family of the Sinapis arvensis genome were cloned and characterized. The 700-bp tandem repeat family is represented by two clones, pSA35 and pSA52, which are 697 and 709 bp in length, respectively. Dot matrix analysis of the sequences indicates the presence of repeated elements within each monomeric unit. Sequence analysis of the repetitive region of clones pSA35 and pSA52 shows that there are several copies of a 7-bp repeat element organized in tandem. The consensus sequence of this repeat element is 5'-TTTAGGG-3'. These elements are highly mutated and the difference in length between the two clones is due to different copy numbers of these elements. The repetitive region of clone pSA35 has 26 copies of the element TTTAGGG, whereas clone pSA52 has 28 copies. The repetitive region in both clones is flanked on either side by inverted repeats that may be footprints of a transposition event. Sequence comparison indicates that the element TTTAGGG is identical to telomeric repeats present in Arabidopsis, maize, tomato, and other plants. However, Bal31 digestion kinetics indicates non-telomeric localization of the 700-bp tandem repeats. The clones represent a novel repeat family as (i) they contain telomere-like motifs as subrepeats within each unit; and (ii) they do not hybridize to related crucifers and are species-specific in nature.

Families of tandemly repeated DNA elements from horse: cloning, nucleotide sequence, and organization

DNA repeats, revealed initially by digestion of horse DNA with restriction enzymes, were cloned and characterized by cross-hybridization studies and nucleotide sequencing. The Sau-like family of tandem repeats contained two classes of repetitive elements with unit repeats of about 80 bp that shared no sequence similarity. Both unit repeats were present, frequently in tandem, in cloned segments of horse DNA of less than 600 bp. Evidence is presented, based on their ladderlike patterns of hybridization to horse DNA and their high level of similarity to published sequences of satellites from equine DNA, suggesting that they are centromerically located in the horse genome. The Sau-like tandem repeat families were specific to Equidae. Another class of repeats, GATA-like elements, which did not appear to have a centromeric distribution, was also cloned and sequenced.

A dot-matrix program with dynamic threshold control suited for genomic DNA and protein sequence analysis

Graphical dot-matrix plots can provide the most complete and detailed comparison of two sequences. Presented here is DOTTER 2, a dot-plot program for X-windows which can compare DNA or protein sequences, and also DNA versus protein. The main novel feature of DOTTER is that the user can vary the stringency cutoff interactively, so that the dot-matrix only needs to be calculated once. This is possible thanks to a ‘Greyramp tool’ that was developed to change the displayed stringency of the matrix by dynamically changing the greyscale rendering of the dots. The Greyramp tool allows the user to interactively change the lower and upper score limit for the greyscale rendering. This allows exploration of the separation between signal and noise, and fine-grained visualization of different score levels in the dot-matrix. Other useful feature are dot-matrix compression, mouse-controlled zooming, sequence alignment display and saving/loading of dot-matrices. Since the matrix only has to be calculated once and since the algorightm is fast and linear in space, DOTTER is practical to use even for sequences as long as cosmids. DOTTER was integrated in the gene-modelling module of the genomic database system ACEDB. This was done via the homology viewer BLIXEM in a way that also allows segments from the BLAST suite of searching programs to be superimposed on top of the full dot-matrix. This feature can also be used for very quick finding of the strongest matches. As examples, we analyse a Caenorhabditis elegans cosmid with several tandem repeat families, and illustrate how DOTTER can improve gene modelling.

Cloning and characterization of the majority of repetitive DNA in cotton (GossypiumL.)

Repetitive DNA elements representing 60-70% of the total repetitive DNA in tetraploid cotton (Gossypium barbadense L.) and comprising 30-36% of the tetraploid cotton genome were isolated from a genomic library of DNA digested with a mixture of four blunt-end cutting restriction enzymes. A total of 313 clones putatively containing nuclear repetitive sequences were classified into 1103 families, based on cross hybridization and Southern blot analysis. The 103 families were characterized in terms of genome organization, methylation pattern, abundance, and DNA variation. As in many other eukaryotic genomes, interspersed repetitive elements are the most abundant class of repetitive DNA in the cotton genome. Paucity of tandem repeat families with high copy numbers (>10(4)) may be a unique feature of the cotton genome as compared with other higher plant genomes. Interspersed repeats tend to be methylated, while tandem repeats seem to be largely unmethylated in the cotton genome. Minimal variation in repertoire and overall copy number of repetitive DNA elements among different tetraploid cotton species is consistent with the hypothesis of a relatively recent origin of tetraploid cottons.

A Family of Complex Tandem DNA Repeats in the Telomeres of Chironomus pallidivittatus

A family of 340-bp tandem telomere-associated DNA repeats is present in 50- to 200-kb blocks in seven of the eight paired chromosome ends in Chironomus pallidivittatus. It consists of four main subfamilies, differing from each other by small clusters of mutations. This differentiation may reflect different functional roles for the repeats. Here we find that one subfamily, D3, is consistently localized most peripherally and extends close to the ends of the chromosomes, as shown by its sensitivity to the exonuclease Bal 31. The amounts of D3 are highly variable between individuals. The repeat characteristic for D3 forms a segment with pronounced dyad symmetry, which in single-strand form would give rise to a hairpin. Evidence from an interspecies comparison suggests that a similar structure is the result of selective forces. Another subfamily, M1, is present more proximally in a subgroup of telomeres characterized by a special kind of repeat variability. Thus, a complex block with three kinds of subfamilies may occupy different M1 telomeres depending on the stock of animals. We conclude that subfamilies are differentially distributed between and within telomeres and are likely to serve different functions.

A family of complex tandem DNA repeats in the telomeres of Chironomus pallidivittatus.

A family of 340-bp tandem telomere-associated DNA repeats is present in 50- to 200-kb blocks in seven of the eight paired chromosome ends in Chironomus pallidivittatus. It consists of four main subfamilies, differing from each other by small clusters of mutations. This differentiation may reflect different functional roles for the repeats. Here we find that one subfamily, D3, is consistently localized most peripherally and extends close to the ends of the chromosomes, as shown by its sensitivity to the exonuclease Bal 31. The amounts of D3 are highly variable between individuals. The repeat characteristic for D3 forms a segment with pronounced dyad symmetry, which in single-strand form would give rise to a hairpin. Evidence from an interspecies comparison suggests that a similar structure is the result of selective forces. Another subfamily, M1, is present more proximally in a subgroup of telomeres characterized by a special kind of repeat variability. Thus, a complex block with three kinds of subfamilies may occupy different M1 telomeres depending on the stock of animals. We conclude that subfamilies are differentially distributed between and within telomeres and are likely to serve different functions.

Nucleotide sequence organisation and analysis of the nuclear ribosomal DNA circle of the protozoan parasite Entamoeba histolytica

We have sequenced the extrachromosomal ribosomal DNA (rDNA) circle of the human protozoan parasite Entamoeba histolytica HM-1:IMSS and present here the complete sequence organisation of the 24.5-kb molecule. Each circle contains two 5.9-kb rDNA transcription units organised as inverted repeats. The regions downstream (3543 bp) and upstream (9216 bp) of the rDNAs contain various families of short tandem repeats. Some of the upstream repeats share extensive sequence homology with the downstream repeats. In addition to the rDNAs themselves, the rDNA circle appears to code for only one other transcript which is 0.7 kb in size as seen in Northern blots. From DNA sequence analysis, no open reading frame could be assigned to the transcript. Extrachromosomal rDNA circles also exist in other E. histolytica strains. Restriction enzyme maps of rDNA circle were constructed from E. histolytica strains 200:NIH, HK-9 and Rahman; and Entamoeba moshkovskii strain Laredo. Striking differences were observed in the organisation of some of them, e.g. the HK-9, Rahman and Laredo circles contained only one rDNA unit and lacked the 0.7-kb transcript sequence. The short repeat sequences upstream and downstream of rDNAs were present in HK-9 and Rahman but absent in Laredo. Circles with one rDNA unit may be derived from those with two units by homologous recombination at direct repeat sequences located upstream and downstream of the two rDNAs.

BEHAB, a new member of the proteoglycan tandem repeat family of hyaluronan-binding proteins that is restricted to the brain.

Hyaluronan (HA) is a ubiquitous component of the extracellular matrix of all tissues. In the mammalian central nervous system (CNS) HA is present throughout development and into adulthood. While the functions of HA are likely to be mediated by HA-binding proteins, no cell or tissue specific HA-binding proteins have been reported. In an effort to characterize the composition of the extracellular matrix of the CNS, we sought to identify neural HA-binding proteins. We report here the isolation and characterization of a cDNA with a high degree of sequence homology to members of the proteoglycan tandem repeat (PTR) family of HA-binding proteins. Unlike other HA-binding proteins, the expression of this cDNA is restricted to the CNS. We propose the name BEHAB, Brain Enriched HyAluronan Binding protein, for this gene. The expression of BEHAB mRNA is developmentally regulated; expression is first detected in the late embryonic period and peaks during the first two postnatal weeks. In the embryo, BEHAB is expressed at highest levels in mitotically active cells. The sequence of BEHAB has long stretches of identity between rat and cat, suggesting that the encoded protein is functionally important. The size and sequence of BEHAB are consistent with the possibility that it could serve a function like link protein, stabilizing interactions between HA and brain proteoglycans. These observations suggest that existence of other tissue specific HA-binding proteins.

Molecular characterization of repetitive DNA sequences from a B chromosome.

In the parasitic wasp Nasonia vitripennis, certain males carry a B chromosome, called PSR (paternal sex ratio), which causes the compaction and subsequent loss of the paternal chromosomes in fertilized eggs. Because Nasonia are haplo-diploid, this leads to the production of all-male broods. Three families (PSR2, PSR18, PSR22) of related, tandemly repetitive DNAs were shown to be present solely on the PSR chromosome. These three families shared two conserved, palindromic DNA sequences, which may play a role in either PSR function or amplification of the tandem arrays. The tandem repeat family NV79 was determined to be present on the PSR chromosome as well as on at least one of the A chromosomes. This shared repeat as well as two repeat families (NV85, NV126) that were localized on the A chromosomes were detected in two sibling species of N. vitripennis. NV79 and NV126 were also found in the more distantly related species, Trichomalopsis dubius.

Role of EBNA-1 in arresting replication forks at the Epstein-Barr virus oriP family of tandem repeats.

The 20-member family of 30-bp tandem repeats located within the oriP region of Epstein-Barr virus (EBV) can act as a transcriptional enhancer in the presence of EBV nuclear antigen 1 (EBNA-1). A replication fork barrier and a termination site of plasmid replication in human B cells is also found within or near the EBV tandem repeats. Within each tandem repeat is a consensus binding sequence for the EBNA-1 protein that is required for extrachromosomal maintenance of oriP-containing plasmids. To investigate the factors that contribute to the arrest of replication forks and termination in the region of the family of repeats, we have used an in vitro replication system in which replication of EBV recombinant plasmids is initiated from the simian virus 40 (SV40) DNA replication origin in the presence of SV40 T antigen and soluble extracts prepared from human cells. The system can support bidirectional replication, initiating from the SV40 DNA origin with termination occurring in a region opposite the origin. Using two-dimensional agarose gel electrophoresis, we observed a barrier to replication forks in the presence of EBNA-1 in the region of the EBV repeats. Termination occurs at or near the tandem repeats in a manner similar to that observed in vivo (T.A. Gahn and C.L. Schildkraut, Cell 58:527-535, 1989). Reducing the number of repeats from 20 to 6 had little effect on the strength of the replication fork barrier. In the absence of EBNA-1, replication forks also arrested at the EBV repeats, but at a much lower efficiency. The addition of competitor DNA containing the EBV family of repeats can almost completely abolish the replication barrier produced in the presence of EBNA-1.