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At least 721 records · Page 40Linked to original sources
PubMed2001

Similar integration but different stability of Alus and LINEs in the human genome.

Alus and LINEs (LINE1) are widespread classes of repeats that are very unevenly distributed in the human genome. The majority of GC-poor LINEs reside in the GC-poor isochores whereas GC-rich Alus are mostly present in GC-rich isochores. The discovery that LINES and Alus share similar target site duplication and a common AT-rich insertion site specificity raised the question as to why these two families of repeats show such a different distribution in the genome. This problem was investigated here by studying the isochore distributions of subfamilies of LINES and Alus characterized by different degrees of divergence from the consensus sequences, and of Alus, LINEs and pseudogenes located on chromosomes 21 and 22. Young Alus are more frequent in the GC-poor part of the genome than old Alus. This suggests that the gradual accumulation of Alus in GC-rich isochores has occurred because of their higher stability in compositionally matching chromosomal regions. Densities of Alus and LINEs increase and decrease, respectively, with increasing GC levels, except for the telomeric regions of the analyzed chromosomes. In addition to LINEs, processed pseudogenes are also more frequent in GC-poor isochores. Finally, the present results on Alu and LINE stability/exclusion predict significant losses of Alu DNA from the GC-poor isochores during evolution, a phenomenon apparently due to negative selection against sequences that differ from the isochore composition.

Alu Elements↗
PubMed2001

An efficient homologous recombination vector pTV(I) contains a hot spot for increased recombinant protein expression in Chinese hamster ovary cells.

We employed reverse genetics to clone a 5.0 kb genomic DNA hot spot HIRPE (hot spot for increased recombinant protein expression) flanking the plasmid integration site from a recombinant Chinese hamster ovary (CHO) cell line. DNA sequence analysis of the 5.0 kb fragment revealed that HIRPE is enriched for repetitive elements, Alu-like sequences and matrix-associated regions that are known to be linked with transcriptionally active regions in a number of mammalian systems. The construction of a homologous recombination vector, pTV1, containing the 5.0 kb HIRPE genomic DNA, a recombinant gene human CTLA4-Ig, and the dhfr gene as a positive selection marker is described. It was observed that the pTV1 vector targeted the CTLA4Ig gene to a preferred locus in the CHO genome contributing to high recombinant gene expression in transfected CHO cells. Preliminary studies suggest that similar to the observation with the parental cell line, pTV1-generated transfectomas that were analyzed appear to harbor an inverted duplication of the genomic DNA at the plasmid integration site.

Abatacept↗
PubMed1998

cDNA cloning of two splice variants of a human copper-containing monoamine oxidase pseudogene containing a dimeric Alu repeat sequence.

Two alternatively spliced transcripts, psiHLAO1 and psiHLAO2, of a copper-containing monoamine oxidase pseudogene have been isolated from a human-liver cDNA library. The larger psiHLAO1 cDNA (2073bp) contains a 5'-flanking segment of 134bp, followed by an apparent open reading frame (ORF) of 1725bp. The deduced amino acid sequence of this ORF (574 residues) shares 81.0% similarity with the 763-residue monoamine oxidase from human placenta (HPAO) (the N-terminal 533 residues of psiHLAO1 share 86.7% similarity with HPAO). The psiHLAO1 ORF is interrupted by an in-frame stop codon corresponding to amino acid 225 and terminates within a type S(a) dimeric Alu repeat sequence. psiHLAO2 appears to be an alternatively spliced variant of psiHLAO1 that has 413 bases of psiHLAO1 excised according to the 'GT-AG' rule. The slightly longer 3' end of the psiHLAO2 transcript shows that the Alu repeat is followed by an 11-bp poly(A) tract that, in turn, is followed by an AT-rich (81%) sequence of 105bp. A reverse transcriptase-polymerase chain reaction (RT-PCR) protocol was used to confirm that both psiHLAO1 and psiHLAO2 are transcribed in human liver and placenta. A search of the expressed sequence tag (EST) database indicates that, like HPAO, psiHLAO derives also from the region 17q21 of the human genome.

Alternative Splicing↗
PubMed1998

CpG doublets, CpG islands and Alu repeats in long human DNA sequences from different isochore families.

A computer analysis of 946 human DNA sequences larger than 50kb and representing about 118Mb of DNA has led to the following observations. (i) Positive correlations hold between CpG levels and the GC levels of isochores and coding sequences, as expected from previous results. (ii) The correlation between CpG levels and the GC levels of pseudogenes is characterized by lower CpG values (at comparable GC levels) and by a lower slope compared with the correlation with coding sequences; this finding suggests that an extensive methylation followed by deamination has taken place on CpG doublets from inactive genes leading to a further CpG shortage. (iii) The frequency of CpG islands in long human sequences increases with increasing GC and almost parallels gene frequency. (iv) The frequency of Alu sequences also increases with increasing GC, but attains a maximum in H2 isochores, in agreement with previous experimental data. (v) The ratio 5mC/CpG (namely, the methylation level over available sites) decreases with increasing GC levels of isochores. This decrease is due only to a small extent to the increase of (unmethylated) CpG islands in GC-rich isochores, and takes place in spite of the increase of strongly methylated Alu sequences in GC-rich isochores; this stresses the much lower relative methylation (5mC/CpG) of single-copy sequences located in GC-rich isochores relative to those located in GC-poor isochores. (vi) CpG levels of Alus and CpG islands are positively correlated with the GC levels of the long sequences in which they are located. (vii) The CpG levels of both Alus and CpG islands increase with their GC levels.

5-Methylcytosine↗
PubMed1999

YAC fragmentation with repetitive and single-copy sequences: detailed physical mapping of the presenilin 1 gene on chromosome 14.

We constructed new LYS2 fragmentation vectors that allow direct acentric and centric fragmentation of yeast artificial chromosomes (YACs) and selection of fragmented YACs in yeast strain AB1380. The fragmentation vectors were used efficiently with repetitive (e.g., Alu), low-copy (e.g., CA-repeats) and single-copy (e.g., exons) sequences. High recombination efficiencies were obtained in fragmenting two different CEPH YACs with the Alu consensus sequence as target sequences for homologous recombination. Analysis of the acentric Alu fragmentation panel of 788H12, containing the presenilin 1 (PSEN1) gene for familial Alzheimer's disease (AD), indicated that high-resolution YAC fragmentation panels covering the entire parent YAC are obtained. Also, marker content analysis of the fragmentation panel indicated that fragmented YACs were propagated stably without rearrangements. The same fragmentation vectors were used efficiently for fragmentation of 788H12 with unique sequences, i.e., exons 3 and 12 of PSEN1 and D14S77, a polymorphic CA repeat, as target sequences. Together, our YAC fragmentation data of 788H12 provided a size estimate for the coding region of PSEN1 of 60kb and a more precise localization of D14S77 at 25kb upstream of PSEN1.

Alu Elements↗
PubMed2001

Increase of homozygosity in centenarians revealed by a new inter-Alu PCR technique.

In the present study a novel inter-Alu PCR technique that allows one to detect inter-individual differences in the genomic regions flanked by Alu repetitive sequences was developed. Two primers complementary to sequences present in different Alu repeats and marked with two different fluorochromes were used in the same PCR reaction, and the PCR products were separated and analyzed by capillary electrophoresis using an automatic sequencer. The method is highly reliable, and three patterns of peaks (QM376-400, QM780-790 and QM480) appeared to be representative for germ-line polymorphisms, as suggested by the results obtained in nine couples of monozygotic twins and four three-generation families. The frequency of these polymorphic peaks was studied in two different age groups (100 young subjects and 69 centenarians). In two out of the three regions (QM376-400 and QM480) a significant increase in homozygote genotypes frequency was observed in centenarians. These counterintuitive results suggest that increased homozygosity contributes to human longevity. This novel inter-Alu PCR approach could represent a valuable tool to identify longevity-associated DNA sequences interspersed throughout human genome, without making any a priori assumption about their nature and function.

Aged↗
PubMed2002

Towards the structure of the mammalian signal recognition particle.

The signal recognition particle (SRP) is a ubiquitous ribonucleoprotein particle involved in the co-translational targeting of proteins to membranes. Crystal structures are now available for three protein-RNA subcomplexes from the SRP, which give insights into fundamental aspects of protein-RNA recognition, the assembly of stable ribonucleoprotein particles and the mechanism of action of the SRP.

Alu Elements↗
PubMed1997

The use of cloned repetitive sequences as hybridization competitors to detect single copy sequences.

To suppress background hybridization due to repetitive sequence elements, competitor Alu containing clones were isolated from a subclone library of human cosmid clone hP3.1. Pre-annealing of the probe-with this competitor increased the signal well above background. In comparison, the addition of the competitor directly to both the prehybridization and hybridization solution was more effective in reducing background. This dramatically increased the signal to noise ratio of the specific hybridization event. Application to fluorescent in situ hybridization (FISH) is readily apparent.

Binding, Competitive↗
PubMed1999

Duplicated regions of AF-4 intron 4 at t(4;11) translocation breakpoints.

BACKGROUND: AF-4 is a common partner gene of MLL. AF-4 breakpoints occur in introns, but most AF-4 introns are uncharacterized. METHODS AND RESULTS: We cloned AF-4 intron 4 and examined the frequency of breakpoints in this intron. The 5.8-kb intron is rich in repeat sequences and was the site of translocation in 3 of 17 leukemias with t(4;11). We cloned the der (11) and der (4) breakpoints and isolated the fusion transcripts in the cell line MV4-11 and in a de novo acute lymphoblastic leukemia (ALL). Both translocations joined MLL intron 6 and AF-4 intron 4. In MV4-11, 249 bases from AF-4 were present in both derivative chromosomes, indicating duplication. In the de novo ALL, duplication of 446 bases from MLL and AF-4 occurred. Reciprocal fusion transcripts were expressed. CONCLUSIONS: Intronic sequence of AF-4 is useful for molecular diagnosis of t(4;11). Duplicated intronic regions suggest staggered chromosomal breakage.

Adolescent↗
PubMed2004

Minimal conditions for exonization of intronic sequences: 5' splice site formation in alu exons.

Alu exonization, which is an evolutionary pathway that creates primate-specific transcriptomic diversity, is a powerful tool for studying alternative-splicing regulation. Through bioinformatic analyses combined with experimental methodology, we identified the mutational changes needed to create functional 5' splice sites in Alu. We revealed a complex mechanism by which the sequence composition of the 5' splice site and its base pairing with the small nuclear RNA U1 govern alternative splicing. We show that in Alu-derived GC introns the strength of the base pairing between U1 snRNA and the 5' splice site controls the skipping/inclusion ratio of alternative splicing. Based on these findings, we identified 7810 Alus within the human genome that are prone to exonization. Mutations in these Alus may cause genetic disorders or contribute to human-specific protein diversity.

5' Untranslated Regions↗
PubMed2004

PCR-generated artificial ribosomal DNAs from premature termination at Alu sequences.

PCR-amplified product may sometimes not correlate with a DNA state in vivo due to formation of recombinant molecules. Here we show that recombinant product can form in vitro on amplifying the region upstream of the rRNA transcription start point in human ribosomal intergenic spacer. These results provide the first information concerning definite Alu sites where premature polymerase termination occurs.

Alu Elements↗
PubMedDate not supplied

Gene-specific chromatin damage in human spermatozoa can be blocked by antioxidants that target mitochondria.

Incubation of gradient purified human spermatozoa, which are routinely maintained in media prior to IVF and intracytoplasmic sperm injection (ICSI), induced DNA strand breaks (up to 89 nicks x 10(-3) bp) and chromatin release. Unlike highly dispersed Alu repeat sequences, the centromeric heterochromatin was much less susceptible to endonuclease attack. In addition to chromatin release, the permeability of the sperm membrane was altered as evidenced by reduced accessibility of sperm nuclei to decondensation factors in mouse embryo extracts. Hybridization of cDNA microarrays with DNA released from spermatozoa revealed a consistent hypersensitivity of certain genes to endogenous cleavage including TP53, VHL (tumour suppressors), BRCA1 (breast cancer), NOS1 (neurotransmitter), PECAM1, FLT1 (angiogenesis) and CDKN1C (cell cycle/imprinted). N-tert-butyl hydroxylamine (NTBH), a derivative of the anti-teratogenic alpha-phenyl-N-t-butyl nitrone (PBN) and synthetic superoxide dismutase (SOD)/catalase mimetics inhibited chromatin release and sustained or dissipated relative mitochondrial membrane potential. Together, these results show a link between the hyperactivation of sperm mitochondria and chromosomal damage of specific genes in vitro, and that the potential risk of disruption of paternally contributed genes can be circumvented by antioxidants which are known to target mitochondria.

Alu Elements↗
PubMed2000

Northwest African distribution of the CD4/Alu microsatellite haplotypes.

We have analysed a linked microsatellite/Alu polymorphism at the CD4 locus (CD4/Alu) in 666 chromosomes from samples of the Iberian Peninsula, Northwest Africa, and West sub-Saharan Africa. The Iberian Peninsula differs from other European populations by its higher levels of haplotype diversity (0.75), and weaker association between the microsatellite allele 90 and Alu(-) chromosomes. These results are explainable by a substantial gene flow from Northwest Africa. Significant geographic clines for the five major haplotypes suggest a south to north migration from sub-Saharan Africa into Northwest Africa. In spite of this, the consistent presence of haplotype 110(-) in this area is congruent with an ancient and autochthonous human presence in Northwest Africa.

Africa South of the Sahara↗
PubMed1999

Localization of mouse type 2 Alu sequence in schistosomes.

Localization of the type 2 Alu sequence (B2), a highly repetitive DNA sequence in the mouse genome, was examined by in situ polymerase chain reaction (in situ PCR) in schistosomes. The signals to the B2 sequence were detected in the cytoplasm of the tegumental membrane and in the nuclei of the mesenchymal, testicular, ovarian and vitelline cells of 8-week Schistosoma japonicum. In contrast, it was difficult to detect any signals of this sequence in 8-week S. mansoni, whereas in 24-week male S. mansoni the signals were observed in the cytoplasm of the tegumental tubercles and in the nuclei of the mesenchymal and testicular cells. On the other hand, in 24-week female S. mansoni the signals were found in the nuclei of the mesenchymal, ovarian and vitelline cells but not found in the tegument. On the contrary, no hybridization band of the B2 sequence was detected in the amplified DNA of 3-week schistosomula of either species. These observations proved that the host DNA sequences existed in restricted schistosome cells and were accumulated in the schistosome body during their development.

Alu Elements↗
PubMed2000

Effect of 3' terminal adenylic acid residue on the uridylation of human small RNAs in vitro and in frog oocytes.

It is known that several small RNAs including human and Xenopus signal recognition particle (SRP) RNA, U2 small nuclear RNA (snRNA) and 7SK RNAs are posttranscriptionally adenylated, whereas U6 snRNA and ribosomal 5S RNA are posttranscriptionally uridylated on their 3' ends. In this study, we provide evidence that a small fraction of U6 snRNA and 5S ribosomal RNA molecules from human as well as Xenopus oocytes contain a single posttranscriptionally added adenylic acid residue on their 3' ends. These data show that U6 snRNA and 5S rRNAs are posttranscriptionally modified on their 3' ends by both uridylation and adenylation. Although the SRP RNA, 7SK RNA, 5S RNA, and U6 snRNA with the uridylic acid residue on their 3' ends were readily uridylated, all these RNAs with posttranscriptionally added adenylic acid residue on their 3' ends were not uridylated in vitro, or when U6 snRNA with 3' A(OH) was injected into Xenopus oocytes. These results show that the presence of a single posttranscriptionally added adenylic acid residue on the 3' end of SRP RNA, U6 snRNA, 5S rRNA, or 7SK RNA prevents 3' uridylation. These data also show that adenylation and uridylation are two competing processes that add nucleotides on the 3' end of some small RNAs and suggest that one of the functions of the 3' adenylation may be to negatively affect the 3' uridylation of small RNAs.

Adenosine Monophosphate↗
PubMed1999

The Alu domain homolog of the yeast signal recognition particle consists of an Srp14p homodimer and a yeast-specific RNA structure.

The mammalian Alu domain of the signal recognition particle (SRP) consists of a heterodimeric protein SRP9/14 and the Alu portion of 7SL RNA and comprises the elongation arrest function of the particle. To define the domain in Saccharomyces cerevisiae SRP that is homologous to the mammalian Alu domain [Alu domain homolog in yeast (Adhy)], we examined the assembly of a yeast protein homologous to mammalian SRP14 (Srp14p) and scR1 RNA. Srp14p binds as a homodimeric complex to the 5' sequences of scR1 RNA. Its minimal binding site consists of 99 nt. (Adhy RNA), comprising a short hairpin structure followed by an extended stem. As in mammalian SRP9/14, the motif UGUAAU present in most SRP RNAs is part of the Srp14p binding sites as shown by footprint and mutagenesis studies. In addition, certain basic amino acid residues conserved between mammalian SRP14 and Srp14p are essential for RNA binding in both proteins. These findings confirm the common ancestry of the yeast and the mammalian components and indicate that Srp14p together with Adhy RNA represents the Alu domain homolog in yeast SRP that may comprise its elongation arrest function. Despite the similarities, Srp14p selectively recognizes only scR1 RNA, revealing substantial changes in RNA-protein recognition as well as in the overall structure of the complex. The alignment of the three yeast SRP RNAs known to date suggests a common structure for the putative elongation arrest domain of all three organisms.

Alu Elements↗
PubMed2000

Region-specific DNA damage by AT-specific DNA-reactive drugs is predicted by drug binding specificity.

Bizelesin and adozelesin are DNA-reactive antitumor drugs that alkylate adenines at the 3' ends of their preferred binding sites [5'T(A/T)(4)A3'and 5'(A/T)(3)(-4)A3', respectively]. We used these drugs to examine the determinants for region-specific damage of human genomic DNA. The distribution of bizelesin binding motifs in several regions analyzed "in silico" correlated well with the experimentally determined lesions in these regions assessed by quantitative polymerase chain reaction (QPCR) stop assay. In contrast to the typically low motif density, clusters of potential bizelesin binding sites were found in the matrix-associated regions (MAR domains) of the c-myc and apolipoprotein B (apoB) genes. Accordingly, lesions induced by bizelesin in these domains (2.13 and 7.06 lesions kbp(-1) microM(-1), respectively) markedly exceeded lesions in bulk DNA (0.87 lesions kbp(-1) microM(-1)) or in regions with typically low motif density (e.g., 0.75 and 0.87 lesions kbp(-1) microM(-1) in a beta-globin gene and c-myc origin of replication regions, respectively). Consistent with the more frequent, less localized adozelesin motif, actual lesions induced by adozelesin exceeded by severalfold lesions by bizelesin in four selected regions (within the c-myc and HPRT loci). Whereas adozelesin is likely to affect similar regions as bizelesin, adozelesin's more promiscuous binding probably compromises its relative specificity for such targets. In contrast, findings for bizelesin provide for the first time a proof of principle that a small molecular weight drug can preferentially damage specific regions in cellular DNA. Targeting of critical repetitive sequences, such as AT-rich MAR domains, which allow for clustering of drug binding motif, can be the paradigm for region specificity of small molecular weight agents.

AT Rich Sequence↗