Showing papers on "Alu element published in 2001"

Methyl-CpG-binding protein 2 represses LINE-1 expression and retrotransposition but not Alu transcription

Abstract: In order to explore the defense mechanism by which retrotransposons are repressed, we assessed the ability of methyl-CpG-binding protein 2, MeCP2, to influence LINE-1 (L1) and Alu transcription and, furthermore, L1 retrotransposition. In transient transfection assays, targeting of the transcriptional-repression domain (TRD) of MeCP2 (via a linked Gal4 DNA-binding domain) to the transcriptional start site of L1 promoter-driven reporter constructs efficiently repressed transcription. The Gal4-linked TRD of the related methyl-CpG-binding protein MBD1 also repressed transcription but not that of MBD2. Furthermore, full-length MeCP2 effectively repressed transcription of a HpaII-methylated L1 reporter. Secondly, we used a genetic assay employing a full-length neo-marked L1 reporter construct to study L1 retrotransposition. We found the Gal4-linked TRD of MeCP2 to repress effectively L1 retrotransposition when targeted to the retrotransposition reporter. Retrotransposition was also reduced in response to in vitro HpaII methylation of the reporter and was further decreased by co-expressed full-length MeCP2. In striking contrast expression of the Gal4-linked TRD of MeCP2 had no inhibiting effect on transcription of an AluSx reporter tagged with a 7S-upstream sequence. Furthermore, full-length MeCP2 abrogated the methylation-induced repression of this reporter. Our results indicate that MeCP2 serves a role in repression of L1 expression and retrotransposition but has no inhibiting effect on Alu transcription.

Patterns of ancestral human diversity: an analysis of Alu-insertion and restriction-site polymorphisms.

Abstract: We have analyzed 35 widely distributed, polymorphic Alu loci in 715 individuals from 31 world populations. The average frequency of Alu insertions (the derived state) is lowest in Africa (.42) but is higher and similar in India (.55), Europe (.56), and Asia (.57). A comparison with 30 restriction-site polymorphisms (RSPs) for which the ancestral state has been determined shows that the frequency of derived RSP alleles is also lower in Africa (.35) than it is in Asia (.45) and in Europe (.46). Neighbor-joining networks based on Alu insertions or RSPs are rooted in Africa and show African populations as separate from other populations, with high statistical support. Correlations between genetic distances based on Alu and nuclear RSPs, short tandem-repeat polymorphisms, and mtDNA, in the same individuals, are high and significant. For the 35 loci, Alu gene diversity and the diversity attributable to population subdivision is highest in Africa but is lower and similar in Europe and Asia. The distribution of ancestral alleles is consistent with an origin of early modern human populations in sub-Saharan Africa, the isolation and preservation of ancestral alleles within Africa, and an expansion out of Africa into Eurasia. This expansion is characterized by increasing frequencies of Alu inserts and by derived RSP alleles with reduced genetic diversity in non-African populations.

Alu Insertion Polymorphisms for the Study of Human Genomic Diversity

Abstract: Genomic database mining has been a very useful aid in the identification and retrieval of recently integrated Alu elements from the human genome. We analyzed Alu elements retrieved from the GenBank database and identified two new Alu subfamilies, Alu Yb9 and Alu Yc2, and further characterized Yc1 subfamily members. Some members of each of the three subfamilies have inserted in the human genome so recently that about a one-third of the analyzed elements are polymorphic for the presence/absence of the Alu repeat in diverse human populations. These newly identified Alu insertion polymorphisms will serve as identical-by-descent genetic markers for the study of human evolution and forensics. Three previously classified Alu Y elements linked with disease belong to the Yc1 subfamily, supporting the retroposition potential of this subfamily and demonstrating that the Alu Y subfamily currently has a very low amplification rate in the human genome.

SINE insertions in cladistic analyses and the phylogenetic affiliations of Tarsius bancanus to other primates.

Abstract: Transpositions of Alu sequences, representing the most abundant primate short interspersed elements (SINE), were evaluated as molecular cladistic markers to analyze the phylogenetic affiliations among the primate infraorders. Altogether 118 human loci, containing intronic Alu elements, were PCR analyzed for the presence of Alu sequences at orthologous sites in each of two strepsirhine, New World and Old World monkey species, Tarsius bancanus, and a nonprimate outgroup. Fourteen size-polymorphic amplification patterns exhibited longer fragments for the anthropoids (New World and Old World monkeys) and T. bancanus whereas shorter fragments were detected for the strepsirhines and the outgroup. From these, subsequent sequence analyses revealed three Alu transpositions, which can be regarded as shared derived molecular characters linking tarsiers and anthropoid primates. Concerning the other loci, scenarios are represented in which different SINE transpositions occurred independently in the same intron on the lineages leading both to the common ancestor of anthropoids and to T. bancanus, albeit at different nucleotide positions. Our results demonstrate the efficiency and possible pitfalls of SINE transpositions used as molecular cladistic markers in tracing back a divergence point in primate evolution over 40 million years old. The three Alu insertions characterized underpin the monophyly of haplorhine primates (Anthropoidea and Tarsioidea) from a novel perspective.

Characterization of the 13q14 tumor suppressor locus in CLL: identification of ALT1, an alternative splice variant of the LEU2 gene.

Abstract: Chromosome 13q14 deletions constitute the most common genetic abnormality in chronic lymphocytic leukemia (CLL). To identify the putative tumor suppressor gene targeted by 13q14 genomic loss, we completely sequenced and characterized a segment of 790 kb at 13q14 spanning the minimal region of loss in CLL. Transcribed sequences in the region were identified through database homology searches and exon-prediction analysis. Two-hundred kb at the centromeric end of the sequence contain five CpG islands, three previously identified genes LEU5/RFP2, LEU2, and LEU1, seven of seven EST clusters composed of >10 ESTs, and a large number of predicted exons. Homology searches against the mouse EST database have allowed us to identify a highly conserved alternative first exon of the LEU2 gene, giving rise to a novel transcript, ALT1 (GenBank accession no. AF380424), which originates within a G+C region in the vicinity of the D13S272 marker. Two novel 3′ exons of LEU2 were also identified and are present in both LEU2 and ALT1 transcripts. However, we have not identified any mutations in leukemia cases, or alterations in expression of mRNAs in the region, that might directly implicate these mRNAs in the pathology of CLL. The centromeric end of the sequence, where all reported genes are located, contains twice the expected amount of ALU repeats, whereas the telomeric end is LINE1 rich and contains four LINE1 elements longer than 4 kb, including two full-length LINE1 sequences. This feature of the sequence may favor the occurrence of chromosomal rearrangements and may confer instability to the region, resulting in deletions that may inactivate an as yet unidentified tumor suppressor.

Alu-mediated inactivation of the human CMP- N-acetylneuraminic acid hydroxylase gene.

Abstract: Inactivation of the CMP-N-acetylneuraminic acid hydroxylase gene has provided an example of human-specific genomic mutation that results in a widespread biochemical difference between human and nonhuman primates. We have found that, although a region containing a 92-bp exon and an AluSq element in the hydroxylase gene is intact in all nonhuman primates examined, the same region in the human genome is replaced by an AluY element that was disseminated at least one million years ago. We propose a mechanistic model for this Alu-mediated replacement event, which deleted the 92-bp exon and thus inactivated the human hydroxylase gene. It is suggested that Alu elements have played potentially important roles in genotypic and phenotypic evolution in the hominid lineage.

Transcriptional activation of short interspersed elements by DNA-damaging agents

Abstract: Short interspersed elements (SINEs), typified by the human Alu repeat, are RNA polymerase III (pol III)-transcribed sequences that replicate within the genome through an RNA intermediate. Replication of SINEs has been extensive in mammalian evolution: an estimated 5% of the human genome consists of Alu repeats. The mechanisms regulating transcription, reverse transcription, and reinsertion of SINE elements in genomic DNA are poorly understood. Here we report that expression of murine SINE transcripts of both the B1 and B2 classes is strongly upregulated after prolonged exposure to cisplatin, etoposide, or gamma radiation. A similar induction of Alu transcripts in human cells occurs under these conditions. This induction is not due to a general upregulation of pol III activity in either species. Genotoxic treatment of murine cells containing an exogenous human Alu element induced Alu transcription. Concomitant with the increased expression of SINEs, an increase in cellular reverse transcriptase was observed after exposure to these same DNA-damaging agents. These findings suggest that genomic damage may be an important activator of SINEs, and that SINE mobility may contribute to secondary malignancy after exposure to DNA-damaging chemotherapy.

Germ line insertion of mtDNA at the breakpoint junction of a reciprocal constitutional translocation.

Abstract: Constitutional chromosomal translocations are relatively common causes of human morbidity, yet the DNA double-strand break (DSB) repair mechanisms that generate them are incompletely understood. We cloned, sequenced and analyzed the breakpoint junctions of a familial constitutional reciprocal translocation t(9;11)(p24;q23). Within the 10-kb region flanking the breakpoints, chromosome 11 had 25% repeat elements, whereas chromosome 9 had 98% repeats, 95% of which were L1-type LINE elements. The breakpoints occurred within an L1-type repeat element at 9p24 and at the 3'-end of an Alu sequence at 11q23. At the breakpoint junction of derivative chromosome 9, we discovered an unusually large 41-bp insertion, which showed 100% identity to 12S mitochondrial DNA (mtDNA) between nucleotides 896 and 936 of the mtDNA sequence. Analysis of the human genome failed to show the preexistence of the inserted sequence at normal chromosomes 9 and 11 breakpoint junctions or elsewhere in the genome, strongly suggesting that the insertion was derived from human mtDNA and captured into the junction during the DSB repair process. To our knowledge, these findings represent the first observation of spontaneous germ line insertion of modern human mtDNA sequences and suggest that DSB repair may play a role in inter-organellar gene transfer in vivo. Our findings also provide evidence for a previously unrecognized insertional mechanism in human, by which non-mobile extra-chromosomal fragments can be inserted into the genome at DSB repair junctions.

HERV-K(OLD): Ancestor Sequences of the Human Endogenous Retrovirus Family HERV-K(HML-2)

Abstract: Sequences homologous to the human endogenous retrovirus (HERV) family HERV-K(HML-2) are present in all Old World primate species. A previous study showed that a central region of the HERV-K(HML-2) gag genes in Hominoidea species displays a 96-bp deletion compared to the gag genes in lower Old World primates. The more ancient HERV-K(HML-2) sequences present in lower Old World primates were apparently not conserved during hominoid evolution, as opposed to the deletion variants. To further clarify the evolutionary origin of the HERV-K(HML-2) family, we screened GenBank with the 96-bp gag-sequence characteristic of lower Old World primates and identified, to date, 10 human sequence entries harboring either full-length or partially deleted proviral structures, probably representing remnants of a more ancient HERV-K(HML-2) variant. The high degree of mutations demonstrates the long-time presence of these HERV-K(OLD) proviruses in the genome. Nevertheless, they still belong to the HML-2 family as deduced from dot matrix and phylogenetic analyses. We estimate, based on the family ages of integrated Alu elements and on long terminal repeat (LTR) divergence data, that the average age of HERV-K(OLD) proviruses is ca. 28 million years, supporting an integration time before the evolutionary split of Hominoidea from lower Old World primates. Analysis of HERV-K(OLD) LTR sequences led to the distinction of two subgroups, both of which cluster with LTRs belonging to an evolutionarily older cluster. Taken together, our data give further insight into the evolutionary history of the HERV-K(HML-2) family during primate evolution.

Comparative sequencing of a multicopy subtelomeric region containing olfactory receptor genes reveals multiple interactions between non-homologous chromosomes.

Abstract: In this study, we assess the evolutionary relationships among different chromosomal copies of a subtelomeric block of sequence. This block contains homology to three olfactory receptor genes and is dispersed on at least 14 different chromosome ends in humans. It is single-copy in non-human primates. We analyzed single nucleotide polymorphisms in two 1 kb subregions and a polymorphic Alu insertion within 181 copies of this block from 12 chromosome ends and found evidence for recent interactions between the subtelomeric regions of non-homologous chromosomes. First, several sequence haplotypes are each present on multiple chromosomes, and several chromosomes each have multiple alleles with divergent haplotypes. Secondly, the observed variation clearly indicates that chromosomes 5q, 8p, 11p and/or 15q have each received the block from at least two different sources by non-homologous exchange. In addition, we observe at least one ectopic gene conversion event. Awareness of such exchange among sequences on non-homologous chromosomes is critical for accurate analysis of these complex and dynamic regions of the genome.

The Basques according to polymorphic Alu insertions

Abstract: Polymorphic Alu insertions provide a set of DNA markers of interest in human population genetics. Approximately 1000-2000 of these insertions have not reached fixation within the human genome. Each one of these polymorphic loci most probably resulted from a unique insertional event, and therefore all individuals possessing the insertion are related by descent not just state. In addition, the direction of mutational change is toward the gain of the Alu element at a particular locus. Therefore, the improved knowledge of both the ancestral state and the direction of mutational change greatly facilitates the analysis of population relationships. As a result, Alu insertion polymorphisms represent a significant tool for population genetic studies. In this study, polymorphic Alu insertions have been employed to ascertain phylogenetic relationships among Basque groups and worldwide populations. The Basques are considered to be a geographic isolate with a unique language and customs. They may be direct descendants of Cro-Magnon enclaves from the upper Paleolithic (38,000 to 10,000 years). The Basques are distributed among narrow valleys in northeastern Spain with little migration between them until recently. This characteristic may have had an effect on allelic frequency distributions. With the aim of studying this possible effect, we have analyzed six autosomal polymorphic Alu loci from four different sites within the Spanish Basque region in order to ascertain any genetic heterogeneity among the Basques. The results are consistent with a lack of homogeneity among these four autochthonous Basque groups.

A deletion/insertion mutation in the BRCA2 gene in a breast cancer family: a possible role of the Alu-polyA tail in the evolution of the deletion.

Abstract: Patients with breast and/or ovarian cancer were screened for gross rearrangements in the BRCA2 gene by Southern hybridization, with exon 10 and a fragment of exon 11 used as probes. One breast cancer patient with a positive family history had a 6.2-kb deletion including exons 12 and 13. The deletion breakpoint in intron 11 was in the 3' polyA tail of an Alu element, where a track of approximately 60 adenine nucleotide residues was inserted. Expansion of the Alu-polyA tail may have resulted from polymerase slippage during replication, representing a novel mechanism in which Alu elements mediate deletion/insertion mutations.

ERCC1: A comparative genomic perspective

Abstract: ERCC1 plays an essential role in the nucleotide excision repair (NER) of DNA. We compare 37 kb of sequence from the ERCC1 region on human chromosome 19q13.3 to the orthologous region on mouse chromosome 7. In addition to showing the conserved gene structure between ERCC1, ASE-1, and their murine counterparts, this genomic comparison reveals a highly conserved 497 bp segment found 5 kb upstream of ERCC1 exon 1 that contains a CpG island and previously unidentified "classical" promoter elements. Additional putative regulatory elements are also found within a conserved LINE-1 (long interspersed nuclear element) sequence 800 bp upstream of exon 1 in both human and mouse. Expressed sequence tag (EST) assemblies for human ERCC1 identified numerous splice variants involving exons 1, 2, 3, 7, 8, and 9 that could affect DNA repair efficiencies of ERCC1. A previously undescribed transcript that reads through exon 9 and utilizes the polyadenylation signal of a neighboring Alu element accounts for nearly half of the total splice variants identified in the human EST database. This transcript would theoretically translate to a larger ERCC1 protein product containing a novel C-terminal end. Overall, approximately 18% of publicly available ERCC1 cDNA sequences were determined to be splice variants, while no variants were found in the mouse. The ability to assess novel transcripts and identify candidate regulatory regions demonstrates the potential utility for a catalogue archiving comparative analyses for all genes involved in DNA repair. Our comparative genomic analysis of ERCC1 can be viewed at http://web.uvic.ca/-bioweb/laj.html.

An Alu Insert as the Cause of a Severe Form of Hemophilia A

Abstract: Alu sequences represent a specific human family of interspersed repetitive DNA, with a copy number in excess of 500,000 within the human genome. Alu repeats are rarely present in protein-coding regions of mature RNA, and only a few Alu insert mutations have been described so far. In this paper we present an Alu retroposition event in a family with a severe form of hemophilia A. The inserted Alu element belonging to the youngest Yb8 subfamily disrupts the reading frame at methionine 1224, exon 14 of the factor VIII gene, leading to a stop codon within the inserted sequence. This observation indicates that the retroposition of Alu elements is a continuing process possibly generating various human genetic defects.

Hierarchical assembly of the Alu domain of the mammalian signal recognition particle.

Abstract: The mammalian signal recognition particle (SRP) catalytically promotes cotranslational translocation of signal sequence containing proteins across the endoplasmic reticulum membrane. While the S-domain of SRP binds the N-terminal signal sequence on the nascent polypeptide, the Alu domain of SRP temporarily interferes with the ribosomal elongation cycle until the translocation pore in the membrane is correctly engaged. Here we present biochemical and biophysical evidence for a hierarchical assembly pathway of the SRP Alu domain. The proteins SRP9 and SRP14 first heterodimerize and then initially bind to the Alu RNA 5' domain. This creates the binding site for the Alu RNA 3' domain. Alu RNA then undergoes a large conformational change with the flexibly linked 3' domain folding back by 180 degrees onto the 5' domain complex to form the final compact Alu ribonucleoprotein particle (Alu RNP). We discuss the possible mechanistic consequences of the likely reversibility of this final step with reference to translational regulation by the SRP Alu domain and with reference to the structurally similar Alu RNP retroposition intermediates derived from Alu elements in genomic DNA.

A Mammalian Gene Evolved from the Integrase Domain of an LTR Retrotransposon

Abstract: FIG. 1.—Summary of the structure and coding sequence of the human Gin-1 gene. Sequences of human cDNAs with accession numbers XMp003947.2 (a putative full-length cDNA), BE502574, AW173201.1, AW950418.1, AI631948.1, and AA766836.1 were used to deduce and confirm these data. The full-length protein is 522 amino acids long. The Gin-1 coding region spans nucleotides 36153–15345 in the genomic clone NTp002663.4. Arrowheads and the numbers above them, respectively, indicate the positions and lengths of introns. Several Alu repeats were detected within the two largest introns. Bold letters indicate the region homologous to the most conserved part of the IN domain, detailed in figure 2 and used to obtain the tree shown in figure 3. Amino acids characteristic of the H2C2 and DDE motifs (Khan et al. 1991) and of the most conserved region of the GPY/F module (Malik and Eickbush 1999) are underlined. Ty3/Gypsy long-terminal-repeat (LTR) retrotransposons are among the best-known transposable elements. They inhabit the genomes of many eukaryotic organisms, such as slime molds, plants, fungi, and animals, including vertebrates (Xiong and Eickbush 1990; Malik and Eickbush 1999; Miller et al. 1999; Marin and Llorens 2000). However, in spite of extensive genomic information, these elements had never been found in mammals. In the process of building a database of integrase domain (IN) sequences, we found an intriguing human sequence very similar to the IN of Ty3/Gypsy elements. It was particularly similar to the IN of the Drosophila melanogaster 412 element (E value 5 10227). The sequence of the human gene, which we called Gypsy integrase-1, or Gin-1, was reconstructed by combining information from genomic and cDNA sequences present in the National Center for Biotechnology Information databases (online at http://www.ncbi.nlm.nih.gov/; sequences in TIGR and Sanger Center databases did not provide additional information). Partial mouse, rat, and cow orthologous cDNAs were also detected. Moreover, an apparently full-length mouse cDNA sequence (accession number AK015243) was also found. However, the corresponding genomic sequences are not yet available for any of these other mammalian species. Figure 1 summarizes the structure and describes the protein encoded by the human Gin-1 gene. It has the characteristic H2C2, DDE, and GPY/F motifs found in many retroviral and retrotransposon integrases (Khan et al. 1991; Malik and Eickbush 1999). Homology to IN of Ty3/Gypsy elements spans the whole protein sequence (fig. 2), strongly suggesting that GIN-1 is also, and exclusively, an integrase. The similarity between the human and mouse genes is the expected similarity for orthologous genes of these two species. Amino acidic identity is 446/552 5 85%, while a comparative analysis of 1,138 mouse/human orthologs estimated an average identity in their coding regions of 86.4% (Makalowski and Boguski 1998). Phylogenetic analyses using IN sequences of the known clades of Ty3/Gypsy elements (Malik and Eickbush 1999), as well as of representative Ty1/Copia elements and retroviruses, confirmed that the putative integrase encoded by Gin-1 is very similar to Mdg1 clade elements IN (this clade so far includes D. melanogaster 412 and Mdg1; Malik and Eickbush 1999). Their sequences form a strongly supported monophyletic group

Molecular description of three macro-deletions and an Alu–Alu recombination-mediated duplication in the HPRT gene in four patients with Lesch-Nyhan disease

Abstract: Mutations in the HPRT gene cause a spectrum of diseases that ranges from hyperuricemia alone to hyperuricemia with profound neurological and behavioral dysfunction. The extreme phenotype is termed Lesch-Nyhan syndrome. In 271 cases in which the germinal HPRT mutation has been characterized, 218 different mutations have been found. Of these, 34 (13%) are large- (macro-) deletions of one exon or greater and four (2%) are partial gene duplications. The deletion breakpoint junctions have been defined for only three of the 34 macro-deletions. The molecular basis of two of the four duplications has been defined. We report here the breakpoint junctions for three new deletion mutations, encompassing exons 4-8 (20033bp), exons 4 and 5 (13307bp) and exons 5 and 6 (9454bp), respectively. The deletion breakpoints were defined by a combination of long polymerase chain reaction (PCR) amplifications, and conventional PCR and DNA sequencing. All three deletions are the result of non-homologous recombinations. A fourth mutation, a duplication of exons 2 and 3, is the result of an Alu-mediated homologous recombination between identical 19bp sequences in introns 3 and 1. In toto, two of three germinal HPRT duplication mutations appear to have been caused by Alu-mediated homologous recombination, while only one of six deletion mutations appears to have resulted from this type of recombination mechanism. The other five deletion mutations resulted from non-homologous recombination. With this admittedly limited number of characterized macro-mutations, Alu-mediated unequal homologous recombinations account for at least 8% (3 of 38) of the macro-alterations and 1% (3 of 271) of the total HPRT germinal mutations.

Topoisomerase-I- and Alu-mediated genomic deletions of the APC gene in familial adenomatous polyposis.

Abstract: Germline mutation in the adenomatous polyposis coli (APC) gene results in familial adenomatous polyposis (FAP), a heritable form of colorectal cancer. We have previously reported two novel mutations that delete exons 11 and 14 of the APC gene, respectively, at the cDNA level without any splice junction defects at the genomic level. We describe here the precise breakpoints of the two mutations and the possible mechanisms leading to the genomic rearrangement. The first rearrangement is most likely a topoisomerase-I-mediated non-homologous recombination resulting in a 2-kb deletion that deletes exon 11 of the APC gene. Both 5' and 3' breakpoints have two topoisomerase I recognition sites and runs of pyrimidines within the 10-bp sequences in their vicinity. Further, the 3' breakpoint has an adenine-thymidine-rich region. This is probably the first report of a topoisomerase-I-mediated germline mutation in a tumor suppressor gene. The second rearrangement is most likely an Alu-Alu homologous recombination resulting in a 6-kb deletion encompassing exon 14. The Alu elements at the 5' and 3' breakpoints include the 26-bp core sequence thought to stimulate recombination. In both rearrangements, partial sequences from the long interspersed nuclear element family are in the vicinity of the breakpoints. Other than serving as markers for regions of DNA damage, their precise role in the recombination events, if any, is unclear. Both deletions result in truncated APC proteins missing the β-catenin- and axin-binding domains, resulting in severe polyposis and cancer.

Structure and origin of a novel dimeric retroposon B1-diD.

Abstract: Here we describe a new short retroposon family of rodents. Like the primate Alu element consisting of two similar monomers, it is dimeric, but the left and right monomers are different and descend from B1 and ID short retroposons, respectively. Such elements (B1-dID) were found in the genomes of Gliridae, Sciuridae, Castoridae, Caviidae, and Hystricidae. Nucleotide sequences of this retroposon can be assigned to several structural variants. Phylogenetic analysis of B1-dID and related sequences suggests a possible scenario of B1-dID evolution in the context of rodent evolution.

Nonrandom distribution of interspersed repeat elements in the BCR and ABL1 genes and its relation to breakpoint cluster regions

Abstract: The Philadelphia translocation, t(9;22)(q34;q11), is the microscopically visible product of recombination between two genes, ABL1 on chromosome 9 and BCR on chromosome 22, and gives rise to a functional hybrid BCR-ABL1 gene with demonstrated leukemogenic properties. Breakpoints in BCR occur mostly within one of two regions: a 5 kb major breakpoint cluster region (M-Bcr) and a larger 35 kb minor breakpoint cluster region (m-Bcr) towards the 3′ end of the first BCR intron. By contrast, breakpoints in ABL1 are reported to occur more widely across a >200 kb region which spans the large first and second introns. The mechanisms that determine preferential breakage sites in BCR, and which cause recombination between BCR and ABL1, are presently unknown. In some cases, Alu repeats have been identified at or near sequenced breakpoint sites in both genes, providing indications, albeit controversial, that they may be relevant. For the present study, we carried out a detailed analysis of genomic BCR and ABL1 sequences to identify, classify, and locate interspersed repeat sequences and to relate their distribution to precisely mapped BCR-ABL1 recombination sites. Our findings confirm that Alu are the most abundant class of repeat in both genes, but that they occupy fewer sites than previously estimated and that they are distributed nonrandomly. r-Scan statistics were applied to provide a measure of repeat distribution and to evaluate extremes in repeat spacing. A significant lack of Alu elements was observed across the major and minor breakpoint cluster regions of BCR and across a 25-kb region showing a high frequency of breakage in ABL1. These findings counter the suggestion that occurrence of Alu at BCR-ABL1 recombination sites is likely by chance because of the high density of Alu in these two genes. Instead, as yet unidentified DNA conformation or nucleotide characteristics peculiar to the preferentially recombining regions, including those Alu elements present within them, more likely influence their fragility. © 2001 Wiley-Liss, Inc.

Sperm nuclear matrix association of the PRM1-->PRM2-->TNP2 domain is independent of Alu methylation.

Abstract: Genes or multigenic chromosomal regions are organized by the nuclear matrix into a series of functionally discrete genic domains. Biophysical analysis of the human chromosome 16p13.13 region has shown that the PRM1→PRM2→TNP2 protamine containing multigenic locus is bounded by two sperm nuclear matrix attachment regions (MAR). This domain exists in a transcriptionally readied or potentiated (i.e. open) chromatin state when associated with the nuclear matrix. The MAR-bounded PRM1→PRM2→TNP2 locus is nestled in an Alu repetitive element dense region. Fluorescence in-situ hybridization, analysis of sperm nuclear matrix/halo preparations showed that the PRM1→PRM2→TNP2 domain specifically localizes to the sperm nuclear matrix. This raised the question of whether nuclear matrix association and gene expression in this locus is mediated by Alu methylation. The methylation status of the various Alu elements contained within the human PRM1→PRM2→TNP2 locus was therefore assayed. The seven Alu elements tested, including those associated with the matrix attachment regions within the PRM1→PRM2→TNP2 locus, were fully methylated in sperm DNA. Conversely, these same Alu repeats were hypomethylated within the erythroleukaemic cell line, K562, which does not express any of the genes from this domain. This study shows that Alu methylation status is independent of attachment of PRM1→PRM2→TNP2 locus to the nuclear matrix and that Alu methylation does not play a leading role in the regulation of this domain.

Transcriptional and translational mechanisms of cytochrome b5 reductase isoenzyme generation in humans.

Abstract: Cytochrome b5 reductase (b5R) is an essential enzyme that exists in soluble and membrane-bound isoforms, each with specific functions. In the rat, the two forms are generated from alternative transcripts differing in the first exons. In contrast, the biogenesis of b5R isoforms in the human is not yet well understood. In the present study we have detected three novel alternative exons, designated 1S, S' and 1B, located between the first alternative exon 1M and the common second exon in the human b5R gene. Accordingly, multiple M-type, S-type and SS'-type and B-type transcripts are generated. All types of human b5R transcript are expressed ubiquitously. An analysis of in vitro translation products demonstrated an alternative use of different AUG initiators resulting in the production of various human b5R protein isoforms. Our results indicate that the organization of the 5' region of the b5R gene is not conserved between rodents and humans. Insertion of Alu elements into the human b5R gene, in particular just upstream of the S/S' region, could be responsible for dynamic events of gene rearrangement during evolution.

Nucleosome formation potential of exons, introns, and Alu repeats.

Abstract: A program for constructing nucleosome formation potential profile was applied for investigation of exons, introns, and repetitive sequences. The program is available at http: //wwwmgs.bionet.nsc.ru/mgs/programs/recon/. We have demonstrated that introns and repetitive sequences exhibit higher nucleosome formation potentials than exons. This fact may be explained by functional saturation of exons with genetic code, hindering the localization of efficient nucleosome positioning sites.

Recent Insertion of an Alu Element Within a Polymorphic Human-Specific Alu Insertion

Abstract: Alu elements are a family of short interspersed repeats that have mobilized throughout primate genomes by retrotransposition over the past 65 Myr of primate evolution (for a review, see Deininger and Batzer 1993). In the human genome, Alu elements exist in copy numbers of approximately 500,000 per haploid genome, representing approximately 5% of the genome, and they may be classified into groups of related subfamily members that share common diagnostic substitutions (Batzer et al. 1996b). The major subfamily branches (J, S, and Y) seem to have appeared at different evolutionary times, with J being older than S, and S being older than Y. Not only have the Alu elements contributed to the evolution of the primate genomes, but they also contribute up to 0.4% of human genetic disease according to Deininger and Batzer (1999). Two main mechanisms may produce human diseases: direct insertions of Alu elements within genes (0.1% of human genetic disease), and unequal homologous recombination events between Alu repeats (0.3% of human genetic disease). Some of the human Alu elements have retroposed so recently that their insertion at a specific location within the human genome remains polymorphic. These polymorphic insertions have been used as genetic markers in human evolution studies due to their particular properties: they are rapid and easy to type, are apparently selectively neutral, and have known ancestral states. The insertion of an Alu element into the human genome is almost certainly a unique event, making any pair of Alu insertion alleles identical by descent and free of homoplasy. The use of these polymorphisms in a worldwide survey of human populations has confirmed the African origin of modern humans (Batzer et al. 1994; Batzer et al. 1996a; Stoneking et al. 1997). One of these polymorphic Alu insertions is the PV92 Alu site that is located in chromosome 16, and it has been proved to be human-specific (Batzer et al. 1994). The PV92 Alu insertion element belongs to the youngest subfamily of Alu sequences, the Alu Y subfamily, and, within that, to the Ya5 subfamily, which is defined by five diagnostic changes relative to the Y consensus (Batzer et al. 1996b). The PV92 Alu insertion is most frequent in Amerindians (Novick et al. 1998) and East Asians (Stoneking et al. 1997), while it has lower frequencies elsewhere.