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Results for “Interspersed Repetitive Sequences”

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PubMed1999

DNA repeats in the human genome.

Repetitive DNA sequences, interspersed throughout the human genome, are capable of forming a wide variety of unusual DNA structures with simple and complex loopfolding patterns. The hairpin formed by the fragile X repeat, (CCG)n, and the bipartite triplex formed by the Friedreich's ataxia repeat, (GAA)n/(TTC)n, show simple loopfolding. On the other hand, the doubly folded hairpin formed by the human centromeric repeat, (AATGG)n, the hairpin G-quartet formed by (TTAGGG)n at the 3' telomere overhang, and the hairpin G-quartet, and hairpin C+.C paired i-motif formed by the insulin minisatellite, [formula: see text] show multiple and complex loopfolding. We have performed high resolution nuclear magnetic resonance (NMR) spectroscopy and in vitro replication to show that unique base-pairing and loopfolding render stability to these unusual structures under physiological conditions. The formation of such stable structures offers a mechanism of unwinding which is advantageous during transcription. For example, the formation of the hairpin G-quartet, and hairpin C+.C paired i-motif upstream of the insulin gene may facilitate transcription. These unusual DNA structures also provide unique 'protein recognition motifs' quite different from a Watson-Crick double helix. For example, the hairpin G-quartet formed by (TTAGGG)n at the 3' telomere overhang is specifically recognized and stabilized by the human repair protein, Ku70/Ku80 hetero-dimer, which may be important in the stability of the telomere. However, the formation of the same unusual DNA structures during replication is likely to cause instability in the lengths of the DNA repeats. If the altered (generally expanded) length enhances the probability of the unusual structure during the next cycle of replication, it further increases the instability of the repeat causing a 'dynamic mutation'. In fact, NMR and in vitro replication studies show that the longer the repeat length the higher is the probability of hairpin formation by the fragile X repeat, (CCG)n. In addition, the hairpin of the fragile X repeat, upstream of the FMR-1 gene, is more susceptible to CpG methylation than its duplex thereby leading to methyl-directed suppression of transcription. Thus, the selective advantage of the unusual structures formed by the DNA repeats in the regulation of gene expression may be offset by the genomic instability caused by the same structures during replication. The repeat number is a critical parameter that helps maintain a balance between the advantage gained from an unusual structure during gene expression and the disadvantage posed by the same structure during replication.

Antigens, Nuclear↗

PubMed1990

"PCR-karyotype" of human chromosomes in somatic cell hybrids.

Amplification of human DNA sequences in 16 monochromosomal somatic cell hybrids containing different human chromosomes were performed by the polymerase chain reaction (PCR) using primer directed at human-specific regions of Alu or L1, the two major classes of interspersed repetitive sequences (IRS-PCR). A chromosome-specific pattern of amplification products was observed on agarose gels run with ethidium bromide, producing a "PCR-karyotype." This simple gel analysis provides a rapid method for identifying and monitoring the human chromosomal content of monochromosomal somatic cell hybrids without conventional cytogenetic analysis. Hybrids containing multiple human chromosome produce complex gel patterns, but identification of chromosome content can be achieved by hybridization of PCR products against a reference panel of monochromosomal or highly reduced hybrids representing each human chromosome. This dot-blot method also enables identification of human marker chromosomes or translocated pieces in hybrids that are not identifiable by cytogenetic methods. These IRS-PCR methods should greatly reduce the need for more laborious cytogenetic, isozyme, and Southern blot characterizations of human-rodent cell hybrids.

Animals↗

PubMed1986

Gene distribution and nucleotide sequence organization in the human genome.

Human DNA was fractionated by centrifugation in Cs2SO4 density gradients containing 3,6-bis(acetatomercurimethyl)dioxane (BAMD). Fractions were investigated in their analytical CsCl profiles and a number of specific sequences were localized in them. The results so obtained led to an improved understanding of the organization of nucleotide sequences in the human genome, as well as to the discovery that a class of DNA having a very high G + C content and not represented in the mouse genome, is particularly rich in genes and interspersed repetitive sequences.

Base Sequence↗

PubMed1985

Cloning of a repeat sequence from human DNA which contains a BamHI site.

A human 1.5 kilobase BamHI repeated DNA fragment has been cloned from a genomic library and subcloned in pBR322. It is part of the human homogeneous main-band DNA, it has properties similar to those of long interspersed repetitive sequences (LINES), and differs from the families of human repeated DNA already described.

Cloning, Molecular↗

PubMed1987

Nucleotide sequence analysis of a cloned DNA fragment from human cells reveals homology to retrotransposons.

During molecular cloning of proviral DNA of human spumaretrovirus, various recombinant clones were established and analyzed. Blot hybridization revealed that one of the recombinant plasmids had the characteristic features of a member of the long interspersed repetitive sequences family. The DNA element was analyzed by restriction mapping and nucleotide sequencing. It showed a high degree of amino acid sequence homology of 54.3% when compared with the 5'-terminal part of the pol gene product of the murine retrotransposon LIMd. The 3' region of the cloned DNA element encodes proteins with an even higher degree of homology of 67.4% in comparison to the corresponding parts of a member of the primate KpnI sequence family.

Amino Acid Sequence↗

PubMed1989

Composite transposable elements in the Xenopus laevis genome.

Members of two related families of transposable elements, Tx1 and Tx2, were isolated from the genome of Xenopus laevis and characterized. In both families, two versions of the elements were found. The smaller version in each family (Tx1d and Tx2d) consisted largely of two types of 400-base-pair tandem internal repeats. These elements had discrete ends and short inverted terminal repeats characteristic of mobile DNAs that are presumed to move via DNA intermediates, e.g., Drosophila P and maize Ac elements. The longer versions (Tx1c and Tx2c) differed from Tx1d and Tx2d by the presence of a 6.9-kilobase-pair internal segment that included two long open reading frames (ORFs). ORF1 had one cysteine-plus-histidine-rich sequence of the type found in retroviral gag proteins. ORF2 showed more substantial homology to retroviral pol genes and particularly to the analogs of pol found in a subclass of mobile DNAs that are supposed retrotransposons, such as mammalian long interspersed repetitive sequences, Drosophila I factors, silkworm R1 elements, and trypanosome Ingi elements. Thus, the Tx1 elements present a paradox by exhibiting features of two classes of mobile DNAs that are thought to have very different modes of transposition. Two possible resolutions are considered: (i) the composite versions are actually made up of two independent elements, one of the retrotransposon class, which has a high degree of specificity for insertion into a target within the other, P-like element; and (ii) the composite elements are intact, autonomous mobile DNAs, in which the pol-like gene product collaborates with the terminal inverted repeats to cause transposition of the entire unit.

Amino Acid Sequence↗

PubMed2000

Anchor polymerase chain reaction display: a high-throughput method to resolve, score, and isolate dimorphic genetic markers based on interspersed repetitive DNA elements.

Genes which confer a disease when mutated, or for which population variability contributes to a quantitative trait such as longevity or disease susceptibility, can be localized in the genetic map by use of an appropriately dense set of polymorphic DNA markers. Here we describe an anchor PCR method for high-throughput genotyping, which can be used to amplify the DNA segments flanking an interspersed repetitive sequence such as a transposon, and to limit the number of product bands per reaction to facilitate marker resolution. We used this method to amplify and display DNA fragments flanking the Tc1 transposable elements from different strains of the nematode Caenorhabditis elegans, varying widely in insert number, and to analyze marker segregation in recombinant inbred lines generated from an interstrain cross. Since essentially all eukaryotic genomes contain abundant interspersed repeat families, many of which are dimorphic (for presence or absence of specific elements) among populations, this method can be used for rapid genotyping and fine-scale chromosomal mapping in many species, including those for which extensive mapping and sequencing data do not yet exist.

Animals↗

PubMed1993

Characterisation of a highly repeated DNA sequence from Mycobacterium bovis.

We report characterisation of a novel repeat sequence from a Mycobacterium bovis genomic library. The highly repeated sequence belongs to a family consisting of a 24 base pair (bp) direct repeat (DR), that appears to be organized into clusters on the chromosome. We classify the 24-bp DR into the group of prokaryotic DNA repeats known as the interspersed repetitive sequence elements. The 24-bp DR will be of potential use as a DNA fingerprinting tool in epidemiological studies of M. bovis.

Base Sequence↗

PubMed2001

Comparative analysis of the promoter regions and transcriptional start sites of mouse Ly49 genes.

Despite numerous studies on the function of Ly49 natural killer cell receptors in the mouse, relatively little is known about how these genes are regulated at the transcriptional level. In the present study, we sequenced and compared 800 bp of the promoter region of nine Ly49 genes from C57B1/6 mice. This comparison showed that there is a high degree of sequence identity between the genes, and also revealed a region which is conserved between the mouse genes and the human Ly49L gene, indicating a potential core promoter region. This analysis also found that Ly49B and H differ from the other genes in having long interspersed repetitive sequence in their promoter region which suggests a gene conversion or rearrangement involving these two genes. In addition, we performed 5' rapid amplification of cDNA ends on four Ly49 genes to localize transcriptional start sites. These experiments showed that the transcriptional initiation sites are heterogeneous for all of the genes examined, and that a large majority of Ly49G transcripts originate from the second exon as well as its first intron. Although potential TATA boxes have been previously identified for some of the genes, we did not find evidence that a majority of transcripts initiate at the expected distance downstream of these boxes. Our data suggest that differences in the location of transcriptional start sites contribute to the observed complexity in receptor repertoire patterns.

Animals↗

PubMedDate not supplied

Technology development at the interface of proteome research and genomics: mapping nonpolymorphic proteins on the physical map of mouse chromosomes.

Data obtained from protein spots by peptide mass fingerprinting are used to identify the corresponding genes in sequence databases. The relevant cDNAs are obtained as clones from the Integrated Molecular Analysis of Genome Expression (I.M.A.G.E.) consortium. Mapping of I.M.A.G.E. clones is performed in two steps: first, cDNA clones are hybridized against a 10-hit genomic mouse bacterial artificial chromosome (BAC) library. Second, interspersed repetitive sequence polymerase chain reaction (IRS-PCR) using a single primer directed against the mouse B1 repeat element is performed on BACs. As each cDNA detects several BACs, and each individual BAC has a 50% chance to recover an IRS-PCR fragment, the majority of cDNAs produce at least a single IRS-PCR fragment. Individual IRS fragments are hybridized against high-density spotted filter grids containing the three-dimensional permutated pools of yeast artificial chromosome (YAC) library resources that are currently being used to construct a physical map of the mouse genome. IRS fragments that hybridize to YAC clones already placed into contigs immediately provide highly precise map positions. This technology therefore is able to draw links between proteins detected by 2-D gel electrophoresis and the corresponding gene loci in the mouse genome.

Animals↗

PubMed1989

Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction.

We report a rapid and sensitive method for the detection of base changes in given sequences of genomic DNA. This technique is based on the facts that specific regions of genomic sequences can be efficently labeled and amplified simultaneously by using labeled substrates in the polymerase chain reaction and that in nondenaturing polyacrylamide gels, the electrophoretic mobility of single-stranded nucleic acid depends not only on its size but also on its sequence. The process does not involve restriction enzyme digestion, blotting, or hybridization to probes. We found that most single base changes in up to 200-base fragments could be detected as mobility shifts. RAS oncogene activation was detected by this technique. We also show that the interspersed repetitive sequences of human, Alu repeats are highly polymorphic.

Base Sequence↗

PubMed1985

Informational content of the echinoderm egg.

The sea urchin egg contains a store of mRNA synthesized during oogenesis but translated only after fertilization, which accounts for a large, rapid increase in the rate of synthesis of largely the same set of proteins synthesized by eggs. Starfish oocytes contain a population of stored maternal mRNA that becomes actively translated upon GVBD and codes for a set of proteins distinct from that synthesized by oocytes. The sequence complexity of RNA in echinoderm eggs is about 3.5 x 10(8) nucleotides, enough to code for about 12,000 different mRNAs averaging 3 kb in length. About 2-4% of the egg RNA functions as mRNA during early embryonic development; most of the sequences are rare, represented in a few thousand copies per egg, but some are considerably more abundant. Many of the stored RNA sequences accumulate during the period of vitellogenesis, which lasts a few weeks. The mechanisms of storage and translational activation of maternal mRNA are not well understood. Histone mRNAs are sequested in the egg pronucleus until first cleavage, but other mRNAs are widely distributed in the cytoplasm. The population of maternal RNA includes many very large molecules having interspersed repetitive sequence transcripts colinear with single-copy sequences. The structural features of much of the cytoplasmic maternal RNA is thus reminiscent of incompletely processed nuclear precursors of mRNA. The functional role of these strange molecules is not understood, but many interesting possibilities have been considered. For instance, they may be segregated into different cell lineages during cleavage and/or they may become translationally activated by selective processing during development. Maternal mRNA appears to be underloaded with ribosomes when translated, possibly because the coding sequences are short relative to the size of the mRNA. Most abundant and many rare mRNA sequences persist during embryonic development. The rare sequence molecules are replaced by newly synthesized RNA, but some abundant maternal transcripts appear to persist throughout embryonic development. Most of the proteins present in the egg do not change significantly in mass during development, but a few decline or accumulate substantially. Together, these observations indicate that much of the information for embryogenesis is stored in the egg, although substantial changes in gene expression occur during development.

Animals↗

PubMed2004

Integrated and sequence-ordered BAC- and YAC-based physical maps for the rat genome.

As part of the effort to sequence the genome of Rattus norvegicus, we constructed a physical map comprised of fingerprinted bacterial artificial chromosome (BAC) clones from the CHORI-230 BAC library. These BAC clones provide approximately 13-fold redundant coverage of the genome and have been assembled into 376 fingerprint contigs. A yeast artificial chromosome (YAC) map was also constructed and aligned with the BAC map via fingerprinted BAC and P1 artificial chromosome clones (PACs) sharing interspersed repetitive sequence markers with the YAC-based physical map. We have annotated 95% of the fingerprint map clones in contigs with coordinates on the version 3.1 rat genome sequence assembly, using BAC-end sequences and in silico mapping methods. These coordinates have allowed anchoring 358 of the 376 fingerprint map contigs onto the sequence assembly. Of these, 324 contigs are anchored to rat genome sequences localized to chromosomes, and 34 contigs are anchored to unlocalized portions of the rat sequence assembly. The remaining 18 contigs, containing 54 clones, still require placement. The fingerprint map is a high-resolution integrative data resource that provides genome-ordered associations among BAC, YAC, and PAC clones and the assembled sequence of the rat genome.

Animals↗

PubMed1992

Degenerate oligonucleotide-primed PCR: general amplification of target DNA by a single degenerate primer.

A version of the polymerase chain reaction (PCR), termed degenerate oligonucleotide-primed PCR (DOP-PCR), which employs oligonucleotides of partially degenerate sequence, has been developed for genome mapping studies. This degeneracy, together with a PCR protocol utilizing a low initial annealing temperature, ensures priming from multiple (e.g., approximately 10(6) in human) evenly dispersed sites within a given genome. Furthermore, as efficient amplification is achieved from the genomes of all species tested using the same primer, the method appears to be species-independent. Thus, for the general amplification of target DNA, DOP-PCR has advantages over interspersed repetitive sequence PCR (IRS-PCR), which relies on the appropriate positioning of species-specific repeat elements. In conjunction with chromosome flow sorting, DOP-PCR has been applied to the characterization of abnormal chromosomes and also to the cloning of new markers for specific chromosome regions. DOP-PCR therefore represents a rapid, efficient, and species-independent technique for general DNA amplification.

Animals↗

PubMed2006

MOM1 mediates DNA-methylation-independent silencing of repetitive sequences in Arabidopsis.

The heterochromatic regions around centromeres of animal and plant chromosomes are composed of tandem repetitive sequences, interspersed with transposons and transposon derivatives. These sequences are largely transcriptionally silent and highly methylated, and are associated with specifically modified histones. Although embedded in heterochromatin, Arabidopsis 5S ribosomal RNA genes are among the most highly transcribed genes. However, some 5S genes are silenced, and we show here that this silencing can be suppressed by a reduction in CG methylation. Importantly, we show that mutation of MORPHEUS' MOLECULE 1 (MOM1) releases 5S repeat silencing independently of chromatin properties, as illustrated by the absence of detectable alteration of DNA and histone H3 methylation patterns. MOM1 also prevents transcription of 180-bp satellite repeats and 106B dispersed repeats but not of transposons. Our results provide evidence that transcription of densely methylated and highly repetitive heterochromatic sequences is controlled by two distinct epigenetic silencing pathways, one dependent on and the other independent of DNA methylation.

ATPases Associated with Diverse Cellular Activitie↗

PubMed2006

CGAT: a comparative genome analysis tool for visualizing alignments in the analysis of complex evolutionary changes between closely related genomes.

BACKGROUND: The recent accumulation of closely related genomic sequences provides a valuable resource for the elucidation of the evolutionary histories of various organisms. However, although numerous alignment calculation and visualization tools have been developed to date, the analysis of complex genomic changes, such as large insertions, deletions, inversions, translocations and duplications, still presents certain difficulties. RESULTS: We have developed a comparative genome analysis tool, named CGAT, which allows detailed comparisons of closely related bacteria-sized genomes mainly through visualizing middle-to-large-scale changes to infer underlying mechanisms. CGAT displays precomputed pairwise genome alignments on both dotplot and alignment viewers with scrolling and zooming functions, and allows users to move along the pre-identified orthologous alignments. Users can place several types of information on this alignment, such as the presence of tandem repeats or interspersed repetitive sequences and changes in G+C contents or codon usage bias, thereby facilitating the interpretation of the observed genomic changes. In addition to displaying precomputed alignments, the viewer can dynamically calculate the alignments between specified regions; this feature is especially useful for examining the alignment boundaries, as these boundaries are often obscure and can vary between programs. Besides the alignment browser functionalities, CGAT also contains an alignment data construction module, which contains various procedures that are commonly used for pre- and post-processing for large-scale alignment calculation, such as the split-and-merge protocol for calculating long alignments, chaining adjacent alignments, and ortholog identification. Indeed, CGAT provides a general framework for the calculation of genome-scale alignments using various existing programs as alignment engines, which allows users to compare the outputs of different alignment programs. Earlier versions of this program have been used successfully in our research to infer the evolutionary history of apparently complex genome changes between closely related eubacteria and archaea. CONCLUSION: CGAT is a practical tool for analyzing complex genomic changes between closely related genomes using existing alignment programs and other sequence analysis tools combined with extensive manual inspection.

Algorithms↗

PubMed2003

Enrichment of gene-coding sequences in maize by genome filtration.

Approximately 80% of the maize genome comprises highly repetitive sequences interspersed with single-copy, gene-rich sequences, and standard genome sequencing strategies are not readily adaptable to this type of genome. Methodologies that enrich for genic sequences might more rapidly generate useful results from complex genomes. Equivalent numbers of clones from maize selected by techniques called methylation filtering and High C0t selection were sequenced to generate approximately 200,000 reads (approximately 132 megabases), which were assembled into contigs. Combination of the two techniques resulted in a sixfold reduction in the effective genome size and a fourfold increase in the gene identification rate in comparison to a nonenriched library.

Chromosomes, Plant↗

PubMed1994

A 2.3-Mb yeast artificial chromosome contig spanning from Gabra3 to G6pd on the mouse X chromosome.

A yeast artificial chromosome (YAC) contig of approximately 2.3 Mb has been constructed for the region of the mouse X chromosome between Gabra3 and G6pd. YACs were isolated from four genomic libraries by PCR or hybridization screening for the loci Gabra3, DXHX1104, F8a, DXHX52, DXBay2, L1cam, and Rsvp. Orientation of YACs was performed by isolation of ends using several PCR-based methods, including an adaptation for mouse YAC analysis of interspersed repetitive sequence (IRS)-vector PCR. YAC ends were mapped on a panel of somatic cell hybrids, in an interspecific backcross, using pulsed-field gel (PFG) electrophoresis or by hybridization to other YACs in the contig. Overlapping YACs were further aligned by restriction mapping using rare-cutter restriction endonucleases, as well as by comparison of IRS-PCR fingerprints. This contig represents the largest one assembled to date in the mouse. It should facilitate the isolation of genes in the region and may aid in the completion of a physical contig for the homologous human region, Xq28.

Animals↗