Members of the Alu family of interspersed, repetitive DNA sequences are in the small circular DNA population of monkey cells grown in culture.
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Animals↗
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Results for “Interspersed Repetitive Sequences”
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Short, interspersed repetitive DNA sequences in prokaryotic genomes.
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Genome, Bacterial↗
Interspersed repetitive DNA sequences of the human genome: are they transposons?
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Cloning, Molecular↗
Genetic fate of recombinant adeno-associated virus vector genomes in muscle.
Recombinant adeno-associated virus (rAAV) vectors are promising human gene transfer vectors, because they mediate long-term gene expression in vivo. The vector DNA form responsible for sustained gene expression has not been clearly defined, but it has been presumed that the vector integrates to some degree and persists in this manner. Using two independent methods, we were unable to identify rAAV integrants in mouse muscle. In the first approach, we were unable to recover host cell-vector DNA junctions from a lambda phage library generated using transduced mouse muscle DNA that contained a high vector copy number. Following this result, we devised a PCR assay based on the principle that integrated rAAV vector sequences could be amplified using primers specific for mouse interspersed repetitive sequences (B1 elements). Using this assay, we analyzed transduced mouse muscle DNA isolated from 6 to 57 weeks after injection and did not detect amplification above background levels. Based on the demonstrated sensitivity of the assay, these results suggested that >99.5% of vector DNA was not integrated. Additional analyses using a novel DNA exonuclease showed that the majority of the rAAV vector DNA in muscle persisted over time as transcriptionally active monomeric and concatameric episomes.
Animals↗
[Reassociation of interspersed moderate DNA repeats].
Reassociation kinetics of the fragments of DNA consisting of interspersed repetitive and non-repetitive nucleotide sequences is considered in this paper. Based on the model, suggested by Gavrilov and Mazo (Mol. biol., 11, 101 1977), which takes into account the random DNA shearing, both reassociation kinetics of the total DNA in the region corresponding to interspersed repeat reassociation and that of the isolated preparation of interspersed repetitive sequences are calculated. In both cases influence of the repeat length on the reassociation rate is demonstrated. The estimation of the repetition frequency of rare repeats from pigeon genome is specified using calculations performed.
Animals↗
Transcriptional "silencer" element in rat repetitive sequences associated with the rat insulin 1 gene locus.
The enhancer elements from either simian virus 40 or murine sarcoma virus activate the expression of a transfected rat insulin 1 (rI1) gene when placed within 2.0 kilobases or less of the rI1 gene cap site. Inclusion of 4.0 kilobases of upstream rI1 sequence, however, results in a substantial reduction in the enhancer-dependent insulin gene expression. These observations suggested that a negative transcriptional regulatory element was present between 2.0 and 4.0 kilobases of the rI1 sequence. To test this notion, we employed a heterologous enhancer-dependent transcription assay in which the simian virus 40 72-base-pair repeat is linked to a human beta-globin gene. Addition of the upstream rI1 element to this system decreased the level of enhancer-dependent beta-globin transcription by a factor of 5 to 15. This rI1 "silencer" element functions in a manner relatively independent of position and orientation and requires a cis-dependent relationship to the transcription unit on which it acts. Thus, the silencer sequence seems to have a number of the characteristics of enhancer elements, and we suggest that it may function by the converse of the enhancer mechanism. The rI1 silencer sequence was identified as a member of a long interspersed rat repetitive family. Thus, a potential role for certain repetitive sequences interspersed throughout the eukaryotic genome may be to regulate gene expression by retaining transcriptional activity within defined domains.
Animals↗
Organization of gene and non-gene sequences in micronuclear DNA of Oxytricha nova.
In order to study the derivation of the macronuclear genome from the micronuclear genome in Oxytricha nova micronuclear DNA was partially digested with EcoRI, size fractionated, and then cloned in the lambda phage Charon 8. Clones were selected a) at random b) by hybridization with macronuclear DNA or c) by hybridization with clones of macronuclear DNA. One group of these clones contains only unique sequence DNA, and all of these had sequences that were homologous to macronuclear sequences. The number of macronuclear genes with sequences homologous to these micronuclear clones indicates that macronuclear sequences are clustered in the micronuclear genome. Many micronuclear clones contain repetitive DNA sequences and hybridize to numerous EcoRI fragments of total micronuclear DNA, yielding similar but non-identical patterns. Some micronuclear clones containing these repetitive sequences also contained unique sequence DNA that hybridized to a macronuclear sequence. These clones define a major interspersed repetitive sequence family in the micronuclear genome that is eliminated during formation of the macronuclear genome.
Animals↗
DNA fingerprinting of pathogenic bacteria by fluorophore-enhanced repetitive sequence-based polymerase chain reaction.
Fluorophore-labeled oligonucleotide primers complementary to defined interspersed repetitive sequences conserved in diverse bacteria were used in the polymerase chain reaction to generate DNA fingerprint patterns from selected pathogenic bacteria. Fluorophore-enhanced, repetitive sequence-based polymerase chain reaction allowed discrimination between unrelated isolates of penicillin-resistant Streptococcus pneumoniae recovered from pediatric patients and Mycobacterium avium cultured from patients with acquired immunodeficiency syndrome. Combinations of oligonucleotide primers labeled with distinct fluorescent dyes enabled simultaneous DNA fingerprinting and Shiga-like toxin gene detection in enterohemorrhagic Escherichia coli isolates. Fluorophore-enhanced, repetitive sequence-based polymerase chain reaction was performed with either purified DNA or intact cells that were lysed during the polymerase chain reaction. Fluorophore-enhanced, repetitive sequence-based polymerase chain reaction successfully combines polymerase chain reaction amplification and fluorescent label detection for DNA fingerprinting of cultured bacterial pathogens.
Base Sequence↗
Studies of a novel repetitive sequence family in the genome of mice.
A new middle repetitive sequence is described in the mouse genome. It has been revealed with a recombinant clone isolated from a Mus musculus BamHI gene library constructed in pBR322 and containing an insertion of 1.73 kb. When digests of genomic DNA were subjected to Southern blot hybridization, using the 1.73-kb insert as probe, we obtained a light smear and discrete bands, indicating a dispersion in the mouse genome of this sequence. This 1.73-kb sequence seems to be a part of a greater repetitive sequence at least 6 kb in length. The sizes of the bands hybridizing with the 1.73-kb insert are similar when compared between different laboratory strains but differ remarkably between the two species M. musculus and Mus caroli. We have shown also a great variation in the copy number of the sequence studied between these two species. When rat DNA is probed with the 1.73-kb insert, no hybridization is observed. Subcloning of the 1.73-kb sequence in three fragments has pointed out that the reiteration was not homogeneous along the 1.73-kb sequence. The 1.73-kb clone was sequenced and compared with other interspersed repetitive sequences, previously described in the rodent genome, and no homology was found.
Animals↗
The human THE-LTR(O) and MstII interspersed repeats are subfamilies of a single widely distributed highly variable repeat family.
Fifteen examples of the transposon-like human element (THE) LTR and thirteen examples of the MstII interspersed repeat are aligned to generate new consensus sequences for these human repetitive elements. The consensus sequences of these elements are very similar, indicating that they compose subfamilies of a single human interspersed repetitive sequence family. Members of this highly polymorphic repeat family have been mapped to at least 11 chromosomes. Seven examples of the THE internal sequence are also aligned to generate a new consensus sequence for this element. Estimates of the abundance of this repetitive sequence family, derived from both hybridization analysis and frequency of occurrence in GenBank, indicate that THE-LTR/MstII sequences are present every 100-3000 kb in human DNA. The widespread occurrence of members of this family makes them useful landmarks, like Alu, L1, and (GT)n repeats, for physical and genetic mapping of human DNA.
Base Sequence↗
Interspersed repetitive and tandemly repetitive sequences are differentially represented in extrachromosomal covalently closed circular DNA of human diploid fibroblasts.
Extrachromosomal covalently closed circular DNA (cccDNA) was isolated from human diploid fibroblasts by alkaline denaturation/renaturation and CsCl-ethidium bromide isopycnic centrifugation. Probing across these gradient fractions showed a higher proportion of cccDNA sequences homologous to the interspersed highly repetitive Alu I and Kpn I sequences than to the human tandemly-repetitive Eco RI (alphoid) DNA. Cloning of these cccDNAs was then carried out following digestion with restriction endonucleases Hind III, Bam HI or Pst I, and ligation into plasmid pBR322. Many isolated recombinant clones were unstable as seen by a high rate of loss over four cycles of antibiotic selection, and frequent plasmid modifications including deletions adjoining the site of insertion. Of 107 cloned sequences which appeared relatively stable, i.e., survived four cycles of antibiotic selection without incurring detectable deletions, 28% and 11% showed homology to Alu I and Kpn I families, respectively, while 4% contained sequences homologous to both. In contrast, less than one percent hybridized to probes for tandemly-repetitive sequences, Eco RI and Satellite III. The average insert size of cloned cccDNA derived from human fibroblasts, 2.52 Kbp, was larger than previously reported for similar clones derived from genetically less stable permanent lines, which may reflect differences in the process of cccDNA generation.
Cell Line↗
DNA sequences of two expressed nuclear genes for human mitochondrial ADP/ATP translocase.
Mitochondrial ADP/ATP translocase is an abundant component of the inner membrane. It carries ATP from the matrix into the intermembrane space and transports ADP back. Clones coding for two different but related forms of the protein have been characterized from bovine cDNA libraries. The corresponding genes are referred to as T1 and T2 and they are expressed at different levels in bovine tissues. The bovine cDNAs have been used to isolate clones from a human genomic library that contain the human T1 and T2 genes. Two nucleotide sequences of 9756 and 8625 base-pairs have been determined and they contain the transcribed regions of the human T1 and T2 genes which cover 4.2 and 5.9 kb of the human genome, respectively (1 kb = 10(3) base-pairs). Both genes are split into four exons. The introns in each gene are at exactly equivalent locations and interrupt sequences coding for segments of the protein that are thought to be extramembranous loops linking transmembrane segments. The proteins encoded in the genes differ in 32 amino acids out of 297, and in common with other ADP/ATP translocases, neither has a processed mitochondrial import sequence. The human T1 and T2 genes are members of a larger gene family that includes a third expressed gene T3 and also at least two spliced pseudogenes. Other studies have shown that T3 is expressed in liver and HeLa cells, and different levels of transcripts of T1 have been found in various tissues. A notable feature of the T1 and T2 genes, that may influence their expression, is that "CpG-rich islands" are associated with their 5' ends. That of the T2 gene contains numerous potential sites for binding the mammalian transcription factor SP1, but no TATA or CCAAT sequences are evident near to its 5' end, although these latter features are associated with the human T1 gene. The two DNA sequence also contain many short interspersed repetitive sequences including 11 Alu repeats, and a novel element about 236 base-pairs in length, which is repeated in a six-fold tandem array in intron B of the T2 gene.
Amino Acid Sequence↗
Repetitive sequence transcripts and U1 RNA in mouse oocytes and eggs.
Others have reported that about two-thirds of the polyadenylated RNA of sea urchin or frog eggs contains short interspersed repetitive sequence transcripts, a much larger proportion than that found in mRNA of somatic cells. Thus, it appears that incompletely processed transcripts accumulate in these oocytes. Also, in what may be a related phenomenon, the nuclear concentration of U1 RNA (involved in processing hnRNA) decreases during growth of frog oocytes. To pursue this question in mammals, Northern blots of RNA from mouse oocytes and eggs collected before and after meiotic maturation were probed with genomic clones containing rodent Alu-equivalent sequences. The Alu sequence is the predominant short interspersed repetitive element in the genome and is abundant in hnRNA. When compared on the basis of mRNA content, the oocyte and egg RNA contained less short repetitive sequence transcripts than liver or brain cytoplasmic RNA. Using a U1 RNA-specific probe, the concentration of U1 RNA in mouse oocyte nuclei was found to be quite similar to that in somatic cells, and U1 RNA was stable during meiotic maturation. These results suggest that processing of transcripts in mouse oocytes does not possess the unusual features observed in lower animals.
Animals↗
Nucleotide sequence of the BALB/c mouse beta-globin complex.
The nucleotide sequence of 55,856 base-pairs containing all seven beta-globin homologous structures from chromosome 7 of the BALB/c mouse is reported. This sequence links together previously published sequences of the beta-globin genes, pseudogenes and repetitive elements. Using low stringency computer searches, we found no additional beta-globin homologous sequences, but did find many more long interspersed repetitive sequences (L1) than predicted by hybridization. L1 is a major component of the mouse beta-globin complex with at least 15 elements comprising about 22% of the reported sequence. Most open reading frames greater than 300 base-pairs in the cluster overlap with L1 repeats or globin genes. Polypurine, polypyrimidine and alternating purine/pyrimidine tracts are not evenly dispersed throughout the complex, but they do not appear to be excluded from or restricted to particular regions. Several regions of intergenic homology were detected in dot-plot comparisons of the mouse sequence with itself and with the human beta-globin sequence. The significance of these homologies is unclear, but these regions are candidates for further study in functional assays in erythroid cell lines or transgenic animals.
Animals↗
The characterization of DINE-1, a short, interspersed repetitive element present on chromosome and in the centric heterochromatin of Drosophila melanogaster.
The banded portion of chromosome 4 (the "dot" chromosome) in Drosophila melanogaster displays some properties of beta-heterochromatin, which is normally found within the centric domain of the chromosomes. The nature and distribution of repetitive elements on chromosome 4 could play a role in the establishment of this unusual chromatin configuration. We describe here one such element: a short, interspersed repetitive sequence named DINE-1. Determination of a consensus sequence for the element reveals that there are two conserved regions (A and B) separated by a highly variable spacer. The conserved sequences are approximately 400 bp long but degenerate at both ends, opening the possibility that a yet-to-be-discovered mother element may be present in the genome. DINE-1 bears few of the properties of the mammalian short interspersed elements (SINEs) to which it bears a superficial resemblance in size. It does not appear to be the product of reverse transcription and lacks any polymerase III promoter consensus. The elements are not flanked by target site duplications and their termini lack direct or inverted repeats, suggesting that they themselves are not transposable. Our analysis of cosmid clones from chromosome 4, and elsewhere in the genome, revealed that the euchromatic locations of DINE-1 are almost exclusively confined to chromosome 4. In situ hybridization of a DINE-1 probe to polytene chromosomes confirmed the preferential distribution along 4, in addition to its presence in the centric heterochromatin. This unusual genomic distribution of bias toward chromosome 4 is also seen in the sibling species, D. simulans, whose dot chromosomes exhibit poorly resolved polytene bands and lack crossing over during meiosis like those of D. melanogaster. However, the dot chromosome of D. virilis, which exhibits a well-defined banded structure on polytene chromosomes and can cross over, has only a single, discrete site of DINE-1 element hybridization. The presence of DINE-1 within these regions suggests a role in the heterochromatic nature of chromosome 4 in D. melanogaster and supports the contention that repeats accumulate in regions of diminished crossing over.
Animals↗
Fluorescence in situ hybridization with Alu and L1 polymerase chain reaction probes for rapid characterization of human chromosomes in hybrid cell lines.
Human-rodent hybrid cell lines have been analyzed with regard to their human DNA content by using various DNA probe sets, derived from the hybrids, for in situ hybridization to normal human metaphase chromosome spreads. Total genomic hybrid DNA was compared with probe sets of hybrid DNA that were highly enriched in human sequences. The latter probes were obtained by amplification through the polymerase chain reaction (PCR) using oligonucleotide primers directed to human specific subsequences of the interspersed repetitive sequences Alu and L1. Previously unidentified chromosomal material within hybrid lines was characterized with speed and precision. It is demonstrated that the complete human complement of hybrid lines can be rapidly assessed by comparing the data obtained with the Alu-PCR products with the results from the L1-PCR products or from the genomic hybrid DNA. This approach using interspersed repetitive sequence-PCR products is simple and fast and also provides an alternative way of generating complex DNA probe sets for the specific delineation of entire chromosomes or subchromosomal regions by in situ hybridization.
Animals↗
Evidence that XR family interspersed RNA may regulate translation in Xenopus oocytes.
It has been shown that about two thirds of Xenopus oocyte or sea urchin egg cytoplasmic poly(A)+ RNA contains interspersed repetitive sequences. The functional significance of this interspersed RNA has remained unknown. Here the function of a subfamily of interspersed RNA (XR family; McGrew and Richter, 1989: Dev Biol 134:267-270) in Xenopus oocytes was studied. We found that the elimination of T7 XR (one of the two complementary strands of the XR repeat) interspersed RNA by complementary oligodeoxynucleotides significantly inhibited protein synthesis. On the other hand, the injection of in vitro synthesized T7 XR RNA stimulated translation. Moreover, the insertion of the T7 XR RNA sequence into globin mRNA repressed the translation of the globin mRNA. In order to explain these results, we analyzed interactions between the XR interspersed RNA and oocyte proteins. We found that the major XR RNA binding proteins were p56 and p60, which could be the known mRNA "masking" proteins that bind mRNA and inhibit translation. Further, a 42 kD protein has been identified that appears to bind T7 XR RNA relatively specifically, although it interacts with mRNA with a lower affinity. Based on all of these data, we have proposed that interspersed RNA may be involved in regulating translation by competing with mRNA to interact with certain proteins that can regulate translation.
Animals↗
Hypomethylation of classical satellite DNA and chromosome instability in lymphoblastoid cell lines.
To determine possible relationships between DNA hypomethylation and chromosome instability, human lymphoblastoid cell lines from different genetic constitutions were studied with regard to 1) uncoiling and rearrangements, which preferentially affect the heterochromatic segments of chromosomes 1 and 16; 2) the methylation status of the tandemly repetitive sequences (classical satellite and alphoid DNAs) from chromosomes 1 and 16, and of the L1Hs interspersed repetitive sequences. The methylation status largely varied from cell line to cell line, but for a given cell line, the degree of methylation was similar for all the repetitive DNAs studied. Two cell lines, one obtained from a Fanconi anemia patient and the other from an ataxia telangiectasia patient were found to be heavily hypomethylated. The heterochromatic segments of their chromosomes 1 and 16 were more frequently elongated and rearranged than those from other cell lines, which were found to be less hypomethylated. Thus, in these lymphoblastoid cell lines, alterations characterized by uncoiling and rearrangements of heterochromatic segments from chromosomes 1 and 16 seem to correlate with the hypomethylation of their repetitive DNAs. Two-color in situ hybridizations demonstrated that these elongations and rearrangements involved only classical satellite-DNA-containing heterochromatin. This specificity may be related to the excess of breakages affecting the chromosomes carrying these structures in a variety of pathological conditions.
Ataxia Telangiectasia↗