Dispersed Repeat Family Research Articles

Ticks are unlikely to play a role in leprosy transmission in the Comoros (East Africa) as they do not harbour M. leprae DNA.

Leprosy, one of the oldest known human diseases, continues to pose a global challenge for disease control due to an incomplete understanding of its transmission pathways. Ticks have been proposed as a potential contributor in leprosy transmission due to their importance as vectors for other infectious diseases. In 2010, a sampling of ticks residing on cattle was conducted on the islands Grande Comore, Anjouan, and Mohéli which constitute the Union of the Comoros where leprosy remains endemic. To investigate the potential role of ticks as a vector in transmission of leprosy disease, molecular analyses were conducted. Out of the 526 ticks analysed, none were found to harbour Mycobacterium leprae DNA, as determined by a quantitative polymerase chain reaction (qPCR) assay targeting a family of dispersed repeats (RLEP) specific to M. leprae. Therefore, our results suggest that in the Union of the Comoros, ticks are an unlikely vector for M. leprae.

Search for Dispersed Repeats in Bacterial Genomes Using an Iterative Procedure

We have developed a de novo method for the identification of dispersed repeats based on the use of random position-weight matrices (PWMs) and an iterative procedure (IP). The created algorithm (IP method) allows detection of dispersed repeats for which the average number of substitutions between any two repeats per nucleotide (x) is less than or equal to 1.5. We have shown that all previously developed methods and algorithms (RED, RECON, and some others) can only find dispersed repeats for x ≤ 1.0. We applied the IP method to find dispersed repeats in the genomes of E. coli and nine other bacterial species. We identify three families of approximately 1.09 × 106, 0.64 × 106, and 0.58 × 106 DNA bases, respectively, constituting almost 50% of the complete E. coli genome. The length of the repeats is in the range of 400 to 600 bp. Other analyzed bacterial genomes contain one to three families of dispersed repeats with a total number of 103 to 6 × 103 copies. The existence of such highly divergent repeats could be associated with the presence of a single-type triplet periodicity in various genes or with the packing of bacterial DNA into a nucleoid.

The Compact Chromatin Structure of a Ty Repeated Sequence Suppresses Recombination Hotspot Activity in Saccharomyces cerevisiae

Recombination between repeated DNA sequences can have drastic consequences on the integrity of the genome. Repeated sequences are abundant in most eukaryotes, yet the mechanism that prevents recombination between them is currently unknown. Ty elements, the main family of dispersed repeats in Saccharomyces cerevisiae, exhibit low levels of exchange. Other regions in the genome have relatively high rates of meiotic recombination (hotspots). We show that a Ty element adjacent to the HIS4 recombination hotspot substantially reduces its activity, eliminating local DSB formation. We demonstrate that the Ty has a closed (nuclease-insensitive) chromatin configuration that is also imposed on the flanking DNA sequences. The compact chromatin structure is determined by sequences at the N terminus of the Ty. Increased binding of the Rap1 protein to the hotspot restores both open chromatin conformation and DSB formation. The chromatin configuration of Ty elements precludes initiation of recombination, thus preventing potentially lethal exchanges between repeated sequences.

Studies on the effects of a flanking repetitive sequence on the expression of single-copy transgenes in Nicotiana sylvestris and in N. sylvestris-N. tomentosiformis hybrids.

To test the influence of a Nicotiana tomentosiformis repetitive sequence (R8.3) on transgene expression in N. sylvestris and in N. sylvestris-N. tomentosiformis hybrids, the R8.3 sequence was placed upstream of a nopaline synthase promoter (NOSpro)-NPTII reporter gene in a T-DNA construct. A number of transgenic N. sylvestris lines were produced and in most, the NPTII gene was expressed. In one line, however, the NPTII gene became silenced and methylated in the NOSpro region. The silenced locus was able to trans-inactivate and induce methylation of two stably expressed transgene loci comprising a similar construct. Nucleotide sequence analyses of the three transgene loci revealed that they each contained a single incomplete copy of the T-DNA, which had sustained deletions of varying sizes in the R8.3 region. Paradoxically, the R8.3 DNA upstream of the two active, unmethylated NOSpro-NPTII genes was highly methylated, whereas the R8.3 DNA upstream of the silenced, methylated NOSpro-NPTII gene was less methylated. The methylated portions of the R8.3 sequence corresponded to retroelement remnants. An active NOSpro-NPTII gene downstream of a nearly intact R8.3 sequence did not become methylated in N. sylvestris-N. tomentosiformis hybrids. Thus, methylation in the R8.3 sequence did not spread into adjoining transgene promoters and the effect of the R8.3 dispersed repeat family on transgene expression was negligible. The silencing phenomena observed with the three single-copy transgene loci are discussed in the context of other possible triggers of silencing.

Repetitive sequences inMycobacterium lepraeand their impact on genome plasticity

About 2% of the genome of Mycobacterium leprae is composed of repetitive DNA. There are more than 26 extinct IS elements together with four families of dispersed repeats, present in five copies or more, RLEP (37 copies), REPLEP (15 copies), LEPREP (eight copies), and LEPRPT (five copies). Although there is no sequence similarity to known transposable elements, RLEP occurs predominantly at the 3'-end of genes and, in several cases, within pseudogenes, suggesting that it was capable of dissemination. Strikingly, on comparison of the genome sequences of M. leprae and the closely related tubercle bacillus, Mycobacterium tuberculosis H37Rv, many of these repetitive sequences were found at sites of discontinuity in gene order. Evidence is presented that loss of synteny, inversion and genome downsizing may have resulted from recombination between dispersed copies of these repetitive elements.

Nucleotide sequence of the partially deleted D4Z4 locus in a patient with FSHD identifies a putative gene within each 3.3 kb element.

Facioscapulohumeral muscular dystrophy (FSHD) is linked to the polymorphic D4Z4 locus on chromosome 4q35. In non-affected individuals, this locus comprises 10-100 tandem copies of members of the 3.3kb dispersed repeat family. Deletions leaving 1-8 such repeats have been associated with FSHD, for which no candidate gene has been identified. We have determined the complete nucleotide sequence of a 13.5kb EcoRI genomic fragment comprising the only two 3.3kb elements left in the affected D4Z4 locus of a patient with FSHD. Sequence analyses demonstrated that the two 3.3kb repeats were identical. They contain a putative promoter that was not previously detected, with a TACAA instead of a TATAA box, and a GC box. Transient expression of a luciferase reporter gene fused to 191bp of this promoter, demonstrated strong activity in transfected human rhabdomyosarcoma TE671 cells that was affected by mutations in the TACAA or GC box. In addition, these 3.3kb repeats include an open reading frame (ORF) starting 149bp downstream from the TACAA box and encoding a 391 residue protein with two homeodomains (DUX4). In-vitro transcription/translation of the ORF in a rabbit reticulocyte lysate yielded two (35)S Cys/ (35)S Met labeled products with apparent molecular weights of 38 and 75kDa on SDS-PAGE, corresponding to the DUX4 monomer and dimer, respectively. In conclusion, we propose that each of the 3.3kb elements in the partially deleted D4Z4 locus could include a DUX4 gene encoding a double homeodomain protein.

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

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

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.

Characterization of species-specific repeated DNA sequences from B. nigra

The construction and characterization of two genome-specific recombinant DNA clones from B. nigra are described. Southern analysis showed that the two clones belong to a dispersed repeat family. They differ from each other in their length, distribution and sequence, though the average GC content is nearly the same (45%). These B genome-specific repeats have been used to analyse the phylogenetic relationships between cultivated and wild species of the family Brassicaceae.

Large size and complex structure of mitochondrial DNA in two nonflowering land plants.

We report the first estimates of genome size and complexity for mitochondrial DNAs (mtDNAs) from nonflowering land plants. The mtDNA of Onoclea sensibilis (sensitive fern) is approximately 300 kb in size, while that of Equisetum arvense (common horsetail) is at least 200 kb. Sufficient mtDNA of Onoclea was available to permit an estimation of the copy number and a linkage analysis of nine mitochondrial genes. Six of these genes appear to be present in only one or two copies in the Onoclea genome, whereas three other genes are present in multiple copies. Five of the approximately ten genes encoding 26S rRNA are located on a large, greater than 10 kb, dispersed repeat that also contains closely linked genes for 18S rRNA and the alpha subunit of ATPase (atpA). The other 26S genes belong to a second dispersed repeat family of greater than 8 kb whose elements do not contain any other identified genes. Because flowering plant mtDNAs are also large and contain dispersed, gene-containing, repeats, it appears that these features arose early in the evolution of land plants, or perhaps even in their green algal ancestors.

A family of dispersed repeats in Mycobacterium leprae

The genome of the causative agent of leprosy, Mycobacterium leprae, contains at least 28 copies of a dispersed repetitive sequence, RLEP. From nucleotide sequence analysis it was clear that the RLEP element consists of a 545 bp central domain flanked by a 100 bp left-end and a 44 bp right-end, sometimes associated with a 47 bp extension. The presence of the left and right ends is variable and this allowed three different RLEP configurations to be defined. When the polymerase chain reaction was used to study variation of the central region at least twelve different classes were detected, suggesting that no two RLEP sequences may be identical. Furthermore, they have few features in common with classical bacterial insertion sequences.

DNA transposition – a major contributor to plant chromosome structure

AbstractHigher plant nuclear genomes contain many families of dispersed repeats that change during evolution. Recent evidence from studies on genetically defined transposable elements raises the possibility that many of the dispersed repeats are remnants of such elements. Transposition of DNA has also occurred between mitochondria, chloroplasts and nuclei, a fact that underlines the major role played by DNA transposition in determining the structure of plant genomes.

DNA sequences and chromosome structure.

In this review evidence for the possible relationship between higher order chromosome structure and the distribution of tandem and dispersed repeated sequences in DNA has been examined. Evidence from studies of chromosome diminution in lower eukaryotes suggests that simple sequence DNAs may have a germ-line function and, in mammals, changes in simple sequence methylation, associated with changes in chromosome condensation, support the idea that the centromeric and telomeric heterochromatin may have a function in germ-line cells. For the major families of dispersed repeats, the weight of available evidence suggests that if they do play a role in chromosome organization, it is not an important one. Cytosine methylation is suggested as a candidate for a role in organizing the chromatin. Long-range patterns of methylation in vertebrates, the relationship of this to gene structure, and the association of changes in methylation with gene activity accord with current evidence linking specific nuclear structures to defined points in coding regions.

Insertion sequences and tandem repetitions as sources of variation in a dispersed repeat family

Sequencing of ten clones from the major dispersed repeat family of mouse (MIF-1) has identified two novel types of sequence variation. Two clones contain small insertion sequences that are flanked by five base-pair duplications of the target site. A third clone, MIFC70, contains a 20 base-pair insertion composed of four direct repeats of a five base-pair sequence. This insertion may have arisen by successive out-of-register pairing and cross-over events at an ancestral target sequence. MIFC70 is also distinguished by its divergence from the standard MIF-1 sequence; whereas for standard clones of the MIF-1 family inter-copy divergence does not exceed 10%, the divergence between MIFC70 and standard clones is of the order of 26%. MIFC70 may represent an aberrant copy which is now isolated from the bulk of the repeat family and is free to diverge.

Discrete and heterogeneous high molecular weight RNAs complementary to a long dispersed repeat family (a possible transposon) of human DNA

Approximately 1% of heterogeneous nuclear RNA and approximately 0.035% of cytoplasmic RNA from a cultured line of human lymphoblastoid cells is complementary to a long dispersed repetitious sequence that comprises at least 6% of human DNA. The complementary nuclear RNA is both heterogeneously and discretely sized and is present in both poly(A)-terminated and non-poly(A)-terminated molecules. The complementary cytoplasmic RNA is mainly in discretely sized molecules ranging in size from approximately 600 to 8200 bases, some of which are most abundantly represented in poly(A)-terminated molecules, whereas others are most abundantly represented in non-poly(A)-terminated molecules. Few, if any, of the complementary cytoplasmic RNAs can be found associated with polyribosomes. The dispersed repeat sequence exhibits substantial restriction enzyme fragment length polymorphisms in human DNA and is also present in mouse DNA, although some regions of the human repeat appear to be more abundantly represented in mouse DNA than are other regions.

The Alu family of dispersed repetitive sequences.

A family of related sequences that includes approximately 500,000 members is the most prominent short dispersed repeat family in primate and rodent DNA's. The primate sequence is approximately 300 base pairs in length and is composed of two imperfectly repeated monomer units, whereas the rodent repeat consists of only a single monomer. Properties of this repeat sequence, its flanking sequences in chromosomal DNA, and RNA's transcribed from it suggest that it may be a mobile DNA element inserted at hundreds of thousands of different chromosomal locations.

Low molecular weight RNAs transcribed in vitro by RNA polymerase III from Alu-type dispersed repeats in Chinese hamster DNA are also found in vivo.

An Alu-type dispersed repeat previously identified in a cloned fragment of Chinese hamster DNA [Haynes, S. R., Toomey, T. P., Leinwand, L. & Jelinek, W. R. (1981) Mol. Cell. Biol. 1, 573-583] serves as a template for cell-free transcription of discrete low molecular weight RNAs by RNA polymerase III [RNA nucleotidyltransferase (DNA-directed), EC 2.7.7.6]. A class of analogous RNAs has been isolated from growing Chinese hamster cells by hybridization of total low molecular weight nuclear RNAs to the cloned DNA fragment from which cell-free transcription occurs. Two-dimensional analysis of RNase digestion products of these RNAs suggests that they are transcribed from multiple members of the Alu-type dispersed repeat family.