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Results for “Interspersed Repetitive Sequences”

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PubMed1996

Use of the polymerase chain reaction to specifically amplify integrated HPV-16 DNA by virtue of its linkage to interspersed repetitive DNA.

A polymerase chain reaction (PCR) based technique that combines a virus specific primer and a human interspersed repetitive sequence (IRS) specific primer in order to detect integration of human papilloma virus type 16 (HPV-16) is described. Amplification of viral-host DNA junctions occurs when viral integration results in placement of the virus specific primer binding site near (less that 3-4 kb) the primer binding site within a repetitive sequence element. The method relies on enzyme labeled oligonucleotide probes to achieve rapid, specific, and nonradioisotopic detection of viral integration related PCR products since episomal forms of the viral DNA do not lead to exponential accumulation of hybridizable PCR products. The technique is demonstrated for human genomic DNA derived from clinical cervical swab specimens and archival paraffin embedded blocks. Viral integration was detected in 41% of the HPV-16 positive samples (n = 34). In this positive subset, 64% were classified as invasive neoplasias, 29% CIN III and 7% CIN II. Analyzing the positive invasive neoplasias, 6 of 9 (66%) of the fingerprint results were obtained when an HPV primer was paired with an Alu primer. Interestingly, 100% of Alu primed fingerprint results obtained were derived from samples presenting invasive neoplasia (P < 0.025 by chi square).

Base Sequence↗

PubMed1999

Molecular differentiation of sex chromosomes probed by comparative genomic hybridization.

Comparative genomic hybridization (CGH) was used to identify and probe sex chromosomes in several XY and WZ systems. Chromosomes were hybridized simultaneously with FluorX-labelled DNA of females and Cy3-labelled DNA of males in the presence of an excess of Cot-1 DNA or unlabelled DNA of the homogametic sex. CGH visualized the molecular differentiation of the X and Y in the house mouse, Mus musculus, and in Drosophila melanogaster: while autosomes were stained equally by both probes, the X and Y chromosomes were stained preferentially by the female-derived or the male-derived probe, respectively. There was no differential staining of the X and Y chromosomes in the fly Megaselia scalaris, indicating an early stage of sex chromosome differentiation in this species. In the human and the house mouse, labelled DNA of males in the presence of unlabelled DNA of females was sufficient to highlight Y chromosomes in mitosis and interphase. In WZ sex chromosome systems, the silkworm Bombyx mori, the flour moth Ephestia kuehniella, and the wax moth Galleria mellonella, the W chromosomes were identified by CGH in mitosis and meiosis. They were conspicuously stained by both female- and male-derived probes, unlike the Z chromosomes, which were preferentially stained by the male-derived probe in E. kuehniella only but were otherwise inconspicuous. The ratio of female:male staining and the pattern of staining along the W chromosomes was species specific. CGH shows that W chromosomes in these species are molecularly well differentiated from the Z chromosomes. The conspicuous binding of the male-derived probe to the W chromosomes is presumably due to an accumulation of common interspersed repetitive sequences.

Animals↗

PubMed1990

The human class I alcohol dehydrogenase gene cluster: three genes are tandemly organized in an 80-kb-long segment of the genome.

The class I alcohol dehydrogenases (ADH; EC 1.1.1.1) play a key role in hepatic alcohol catabolism. Three human class I ADH genes, ADH1, ADH2, and ADH3, which encode the alpha, beta, and gamma subunits respectively, have been isolated and mapped on chromosome 4q21-q23. Genomic cloning using a cosmid vector allowed us to obtain an 88-kb-long genomic segment, which was found to include an entire 80 kb of the class I ADH gene cluster. All three genes lie in the same transcriptional orientation and the order of genes is 5'-ADH3-ADH2-ADH1-3'. It may be of some significance that the order of transcriptional activation in the hepatic development, alpha----beta----gamma, is opposite to the order of gene arrangement. Several members of the AluI family and the KpnI (L1) family of interspersed repetitive sequences were mapped in this region. The divergence of insertional sites suggested that gene multiplication of the class I ADH genes had taken place in the earlier stages of human (or primate) evolution.

Alcohol Dehydrogenase↗

PubMed1992

Allele frequency distribution of the (TG)n(AG)m microsatellite in the apolipoprotein C-II gene.

The dinucleotide (TG)n interspersed repetitive sequences are the most abundant microsatellites in the human genome. Using the polymerase chain reaction to amplify a (TG)n(AG)m microsatellite in the first intron of the apo C-II gene, we have detected 15 different alleles in 242 unrelated individuals of French ancestry. The heterozygosity index was 0.85 and codominant Mendelian inheritance of the alleles was observed in individuals from 121 nuclear families. We report that polymorphism at this locus is attributable to length variation at both (TG)n and (AG)m motifs, although the (AG)m motif contains only two alleles differing by one repeat unit. A quadrimodal allele frequency distribution was observed at the (TG)n(AG)m locus. Each of the first three modes comprises one frequent allele and one very rare allele adjacent in size. No alleles of intermediate size were found between the three first modes. The fourth mode encompasses nine alleles that span from 27 to 35 repeat units. We suggest that this distribution reflects the molecular mechanisms by which alleles give rise to one another.

Alleles↗

PubMed2004

Inaccurate reconstruction of ancestral GC levels creates a "vanishing isochores" effect.

It has recently been proposed, based on an analysis of orthologous gene sequences from closely related species, that GC-rich regions of primate and cetartiodactyl genomes are becoming GC-poorer, i.e., that GC-rich isochores are now vanishing in these lineages. We review an artefact of parsimony-based ancestor reconstruction in GC-rich DNA, and show that its magnitude explains the apparent vanishing of the GC-richest regions in cetartiodactyls, even if they are in fact at compositional equilibrium. The presently available data do not allow the disequilibrium hypothesis to be entirely ruled out in primates, yet, as we argue here, second-order artefacts can accumulate. They are therefore likely to explain many if not all of the observations, rendering unnecessary the general hypothesis of vanishing GC-rich isochores in mammals.

Animals↗

PubMed2000

Genomic scrap yard: how genomes utilize all that junk.

Interspersed repetitive sequences are major components of eukaryotic genomes. Repetitive elements comprise over 50% of the mammalian genome. Because the specific function of these elements remains to be defined and because of their unusual 'behaviour' in the genome, they are often quoted as a selfish or junk DNA. Our view of the entire phenomenon of repetitive elements has to now be revised in the light of data on their biology and evolution, especially in the light of what we know about the retroposons. I would like to argue that even if we cannot define the specific function of these elements, we still can show that they are not useless pieces of the genomes. The repetitive elements interact with the surrounding sequences and nearby genes. They may serve as recombination hot spots or acquire specific cellular functions such as RNA transcription control or even become part of protein coding regions. Finally, they provide very efficient mechanism for genomic shuffling. As such, repetitive elements should be called genomic scrap yard rather than junk DNA. Tables listing examples of recruited (exapted) transposable elements are available at http://www.ncbi.nlm.gov/Makalowski/ScrapYard/

Animals↗

PubMed1992

Isolation of DNA markers from a region between incontinentia pigmenti 1 (IP1) X-chromosomal translocation breakpoints by a comparative PCR analysis of a radiation hybrid subclone mapping panel.

A strategy based on the use of human-specific interspersed repetitive sequence (IRS)-PCR amplification was used to isolate regional DNA markers in the vicinity of the incontinentia pigmenti 1 (IP1) locus. A radiation hybrid (RH) resulting from a fusion of an irradiated X-only somatic cell hybrid (C12D) and a thymidine kinase deficient (TK-) hamster cell line (a23) was identified as containing multiple X chromosome fragments, including DNA markers spanning IP1 X-chromosomal translocation breakpoints within region Xp11.21. From this RH, a panel of subclones was constructed and analyzed by IRS-PCR amplification to (a) identify subclones containing a reduced number of X chromosome fragments spanning the IP1 breakpoints and (b) construct a mapping panel to assist in identifying regional DNA markers in the vicinity of the IP1 locus. By using this strategy, we have isolated three different IRS-PCR amplification products that map to a region between IP1 X chromosome translocation breakpoints. A total of nine DNA sequences have now been mapped to this region; using these DNA markers for PFGE analyses, we obtained a probe order DXS14-DXS422-MTHFDL1-DXS705. These DNA markers provide a starting point for identifying overlapping genomic sequences spanning the IP1 translocation breakpoints; the availability of IP1 translocation breakpoints should assist the molecular analysis of this locus.

Animals↗

PubMed1993

Single-strand conformational polymorphism (SSCP) mapping of the mouse genome: integration of the SSCP, microsatellite, and gene maps of mouse chromosome 1.

Interspersed repetitive sequence (IRS) PCR and repetitive element-to-bubble (IRS-bubble) PCR have been utilized to rapidly generate large numbers of mouse-specific, chromosome 1-enriched STSs from mouse-hamster somatic cell hybrids. Single-strand conformational polymorphism (SSCP) has been used to localize 39 new repetitive element-linked STSs to the mouse map: 22 to Chr 1, 10 to Chr. 15, 2 each to Chrs 12 and 14, and three to Chr 7. In addition, we have integrated the SSCP, single-strand length polymorphism, and restriction fragment length polymorphism maps of mouse Chr. 1, resulting in a high-density map of the chromosome, containing over 100 loci, all typed on a single interspecific backcross.

Animals↗

PubMed2001

Applications of retrotransposons as genetic tools in plant biology.

Retrotransposons are mobile genetic elements that accomplish transposition via an RNA intermediate that is reverse transcribed before integration into a new location within the host genome. They are ubiquitous in eukaryotic organisms and constitute a major portion of the nuclear genome (often more than half of the total DNA) in plants. Furthermore, they are dispersed as interspersed repetitive sequences throughout most of the length of all host chromosomes. These unique properties of retrotransposons have been exploited as genetic tools for plant genome analysis. Major applications are in determining phylogeny and genetic diversity and in the functional analyses of genes in plants. Here, recent advances in molecular markers, gene tagging and functional genomics technologies using plant retrotransposons are described.

Genetic Linkage↗

PubMedDate not supplied

Removal of the Alu structural domain from signal recognition particle leaves its protein translocation activity intact.

Alu-like elements comprise the most abundant family of interspersed repetitive sequences in primates and rodents, and contain many features of processed genes, suggesting that they were initially derived by reverse transcription of processed RNA transcripts. Transcripts containing Alu family members are represented in heterologous nuclear RNAs, cytoplasmic messenger RNAs and small RNAs, although nothing is known about their function. Evolutionary studies strongly suggest that the parent RNA for the Alu-like elements is the highly conserved 7SL RNA, which is an essential component of signal recognition particle (SRP), a small cytoplasmic ribonucleoprotein whose function is the targeting of nascent secretory and membrane proteins to the rough endoplasmic reticulum (for a review see ref. 6). 7SL RNA is composed of both unique and Alu-like sequences. SRP is rod-shaped and, in addition to its RNA, contains four proteins (two monomers composed of a polypeptide of relative molecular mass (Mr) 19,000 (19K) and one of 54K, and two heterodimers, one composed of a 9K and a 14K polypeptide, and the other composed of a 68K and a 72K polypeptide, respectively). The RNA moiety is required for SRP activity, as well as for structural integrity of the particle. To investigate whether the Alu-like segments of 7SL RNA have a specific role in SRP activity, we have now purified and analysed a SRP subparticle that is created upon extensive digestion with micrococcal nuclease and entirely lacks the Alu-like sequences. We find that it contains the 72/68K, 54K and 19K proteins tightly bound, but lacks the 9/14K protein. In vitro activity assays demonstrated that the subparticle could still promote secretory protein translocation across the microsomal membrane, but could no longer trigger an arrest of pre-secretory protein synthesis. Re-addition of the 9/14K protein did not restore the elongation arrest. We conclude that the region of SRP comprised of the Alu-like RNA and the 9/14K protein exists in a distinct structural domain which is not required for the protein translocation promoted by SRP but apparently confers elongation-arresting activity on the particle.

Humans↗

PubMed1992

Localization of a DNA repair gene (XRCC5) involved in double-strand-break rejoining to human chromosome 2.

Complementation of the repair defect in hamster xrs mutants has been achieved by transfer of human chromosome 2 using the method of microcell-mediated chromosome transfer. The xrs mutants belong to ionizing radiation complementation group 5, are highly sensitive to ionizing radiation, and have an impaired ability to rejoin radiation-induced DNA double-strand breaks. Both phenotypes were corrected by chromosome 2, although the correction of radiation sensitivity was only partial. Complementation was achieved in two members of this complementation group, xrs6 and XR-V15B, derived independently from the CHO and V79 cell lines, respectively. The presence of human chromosome 2 in complemented clones was examined cytogenetically and by PCR analysis with primers directed at a human-specific long interspersed repetitive sequence or chromosome 2-specific genes. Complementation was observed in 25/27 hybrids, one of which contained only the q arm of chromosome 2. The two noncomplementing hybrids were missing segments of chromosome 2. The use of a back-selection system enabled the isolation of clones that had lost the human chromosome and these regained radiation sensitivity. Transfer of several other human chromosomes did not result in complementation of the repair defect in XR-V15B. These data show that the gene defective in xrs cells, XRCC5, which is involved in double-strand break rejoining, is located on human chromosome 2q.

Chromosome Banding↗

PubMed1989

High-mobility group protein HMG-I localizes to G/Q- and C-bands of human and mouse chromosomes.

Mammalian metaphase chromosomes can be identified by their characteristic banding pattern when stained with Giemsa dye after brief proteolytic digestion. The resulting G-bands are known to contain regions of DNA enriched in A/T residues and to be the principal location for the L1 (or Kpn 1) family of long interspersed repetitive sequences in human chromosomes. Here we report that antibodies raised against a highly purified and biochemically well characterized nonhistone "High-Mobility Group" protein, HMG-I, specifically localize this protein to the G-bands in mammalian metaphase chromosomes. In some preparations in which chromosomes are highly condensed, HMG-I appears to be located at the centromere and/or telomere regions of mammalian chromosomes as well. To our knowledge, this is the first well-characterized mammalian protein that localizes primarily to G-band regions of chromosomes.

Animals↗

PubMed1996

Spontaneous apoptosis in mouse F4N-S erythroleukemia cells induces a nonrandom fragmentation of DNA.

This study characterizes the fragmented DNA of mouse erythroleukemia (MEL) cells spontaneously entering apoptosis. Fragmented DNA was isolated by introducing a novel procedure that ensured a complete extraction of the characteristic oligonucleosomal ladder. As the results show, less than 10% of DNA of apoptotic cells is fragmented in this form. The main conclusion from experiments to characterize the nature of fragmented DNA is that spontaneous apoptosis induces a nonrandom cleavage of genomic DNA. The Southern analysis performed with total apoptotic DNA revealed that it is strongly enriched in interspersed repetitive sequences. In situ hybridizations with such DNA showed further than in interphase nuclei these sequences flock together and form clusters spread throughout the whole nuclear area whereas in mitotic chromosomes they are located predominantly at their pericentromeric/peritelomeric ends. Partial cloning and sequencing reinforces the notion that the apoptotic DNA is representative for a heterochromatinic portion of the mouse genome. Support for such an unexpected conclusion is coming also from experiments indicating that this DNA is heavily methylated and poorly transcribed.

Animals↗

PubMed1992

Identification of an internal cis-element essential for the human L1 transcription and a nuclear factor(s) binding to the element.

L1 (LINE-1) is a long interspersed repetitive sequence derived from a retrotransposon. Transfection studies using the CAT gene as a reporter demonstrated that the first 155bp in the human L1 sequence contains an element(s) responsible for the promoter activity in HeLa cells. The transcription was shown to initiate at the first nucleotide of the L1 sequence in the transgene. Three prominent nuclear protein binding sites were found in the 5' region of the L1 sequence by DNaseI footprint analysis. One of the binding sites, designated as site A located at +3 to +26, was shown to be essential for the L1 transcription because the mutation at the site A caused almost complete loss of the promoter activity. A sequence AAGATGGCC at +11 to +19 in the site A was defined as a target core element for the protein binding. The site A-binding protein (designated TFL1-A) was found in various types of cells including an embryonic teratocarcinoma cell line. These results indicate that an internal short element located at the very 5' terminal of L1 sequence and the nuclear factor binding to the element play a crucial role in the transcription of human L1.

Amino Acid Sequence↗

PubMed1986

An analysis of replacement and synonymous changes in the rodent L1 repeat family.

L1 is a family of long interspersed repetitive sequences in mammals that includes the BamHI family in rodents and the KpnI family in primates. Previous studies have shown that L1 repeats contain a long open reading frame and that the family evolves in concert. Working with 32 rodent elements for which DNA sequence is available, we used the distribution of replacement and synonymous changes to determine which L1 lineages had been expressing their reading frame. The evidence obtained is consistent with there having been a small number of L1 genes that have been expressing a functional protein. Much of the concerted evolution in L1 is accounted for by the tendency of these functioning L1 genes to continually create nonfunctional pseudogenes by reinsertion into the genome of sequences derived from their transcripts. The gain of new pseudogenes is balanced by the loss of old pseudogenes with a half-life of 2 Myr. Therefore, most of the observed L1 repeats are at a dead end with respect to either the expression of the L1 protein or the potential to elaborate further copies of themselves. However, the turnover of L1 pseudogenes is sufficient to constitute a vast flux of sequences into and then out of the flanking regions of all cellular genes. If the presence of flanking L1 pseudogenes affects the expression of other genes in even a subtle fashion, this process should represent a major source of genetic variation. A second level of concerted evolution occurs within the functional L1 sequences in a pattern that did not meet our expectations for selfish DNA. Also, in spite of the marked suppression of replacement relative to synonymous changes in functioning L1 genes, they evolve at an overall rate accelerated to the level of their own pseudogenes.

Animals↗

PubMed2001

Advanced integrated mouse YAC map including BAC framework.

Functional characterization of the mouse genome requires the availability of a comprehensive physical map to obtain molecular access to chromosomal regions of interest. Positional cloning remains a crucial way of linking phenotype with particular genes. A key step and frequent stumbling block in positional cloning is making a contig of a genetically defined candidate region. The most efficient first step is isolating YAC (Yeast Artificial Chromosome) clones. A robust, detailed YAC contig map is thus an important tool. Employing Interspersed Repetitive Sequence (IRS)-PCR genomics, we have generated an advanced second-generation YAC contig map of the mouse genome that doubles both the depth of clones and the density of markers available. In addition to the primarily YAC-based map, we located 1942 BAC (Bacterial Artificial Chromosome) clones. This allows us to present for the first time a dense framework of BACs spanning the genome of the mouse, which, for instance, can serve as a nucleus for genomic sequencing. Four large-insert mouse YAC libraries from three different strains are included in our data, and our analysis incorporates the data of Hunter et al. and Nusbaum et al. There is a total of 20,205 markers on the final map, 12,033 from our own data, and a total of 56,093 YACs, of which 44,401 are positive for more than one marker.

Algorithms↗

PubMed1996

Mouse euchromatin specific "genome-painting" with a LINE probe: a rapid method for identification and mapping of human chromosomes in mouse-human microcell hybrids by two-color FISH.

We describe the use of a long interspersed repetitive sequence (mCPE1.51) for mouse euchromatin specific "genome-painting". In fluorescence in situ hybridization (FISH) experiments, this probe was suitable for identification of the mouse genome and disclosure of translocations of mouse chromosome segments to chromosomes of different species without suppression hybridization. The euchromatin specificity of the probe allowed the discrimination between euchromatin and heterochromatin of mouse chromosomes. Simultaneous hybridization of the biotinylated mouse specific genome-painting probe and a digoxigenin-labeled human chromosome 3-specific cosmid probe to metaphase spreads of mouse-human microcell hybrid carrying a single deleted human chromosome 3 on a mouse fibrosarcoma background, allowed rapid identification and mapping of human chromosome 3.

Animals↗

PubMed1998

Construction of a whole-genome radiation hybrid panel for high-resolution gene mapping in pigs.

We have developed a panel of 152 whole-genome radiation hybrids by fusing irradiated diploid pig lymphocytes or fibroblasts with recipient hamster permanent cells. The number and size of the porcine chromosome fragments retained in each hybrid clone were checked by fluorescence in situ hybridization with a SINE probe or by primed in situ labeling (PRINS) with SINE-specific primers. A strategy based on the interspersed repetitive sequence polymerase chain reaction (IRS-PCR) was developed for selected clones to determine if the large fragments painted by the SINE probe corresponded to one pig chromosome or to different fragments of several chromosomes. This strategy was buttressed by a double PRINS approach using primers specific for alpha-satellite sequences of two different groups of swine chromosomes. Genome retention frequency was estimated for each clone by PCR with 32 markers localized on different porcine chromosomes. Of the 152 hybrids produced, 126 were selected on the basis of cytogenetic content and chromosome retention frequency to construct a radiation hybrid map of swine chromosome 8. Our initial results for this chromosome indicate that the resolution of the radiation hybrid map is 18 times higher than that obtained by linkage analysis.

Animals↗