Human Alu Sequences Research Articles

The Mre11 Complex Is Required for Repair of Hairpin-Capped Double-Strand Breaks and Prevention of Chromosome Rearrangements

Inverted repeats (IRs) that can form a hairpin or cruciform structure are common in the human genome and may be sources of instability. An IR involving the human Alu sequence ( Alu-IR) has been studied as a model of such structures in yeast. We found that an Alu-IR is a mitotic recombination hotspot requiring MRE11/RAD50/XRS2 and SAE2. Using a newly developed approach for mapping rare double-strand breaks (DSBs), we established that induction of recombination results from breaks that are terminated by hairpins. Failure of the mre11, rad50, xrs2, and sae2 mutants to process the hairpins blocks recombinational repair of the DSBs and leads to generation of chromosome inverted duplications. Our results suggest an additional role for the Mre11 complex in maintaining genome stability.

Quantification of human Alu sequences by real-time PCR--an improved method to measure therapeutic efficacy of anti-metastatic drugs in human xenotransplants.

For measuring the efficacy of new anti-metastatic drugs in preclinical models, macroscopical analysis or classical histology of secondary organs are established methods. However, macroscopical evaluation does not take into consideration intra-organ metastasis. Histological analysis is often performed in few sections of the relevant organs, and this may be misleading, since equal distribution of tumor cells within an organ is unlikely. In addition, recent studies have demonstrated that anti-tumorigenic drugs are able to promote metastasis and to change the metastatic pattern. Therefore, extensive analysis of metastasis is mandatory for the evaluation of new compounds. A feasibility study was conducted to find out if the quantification of human Alu sequences could be applied as a surrogate marker for metastasis in xenografts. Alu PCR was performed by using the LightCycler system, which allows PCR reaction and subsequent quantification of the PCR products in less than 30 min. We found that i) the equivalent of one human tumor cell in 1 x 10(6) murine cells could be detected; ii) in tumor-carrying mice, Alu signal increased over time in secondary organs; iii) this increase was more prominent using highly metastatic tumor cells; iv) Alu signal intensity in DNA extracted from tissue slides correlated with the expression of histological tumor markers; v) in three different tumor models (colon, breast and lung), treatment with Taxol or 5-fluorouracil reduced the amount of Alu in different organs. In contrast, reduction of Alu by the matrix metalloproteinase inhibitor RO 28-2653 was not significant. Taken together, quantification of Alu sequences is a fast and accurate method to evaluate the therapeutic efficacy of anti-metastatic drugs in xenografts.

PRINS, the other in situ DNA labeling method useful in cellular biology

PRINS has proven to be an attractive alternative to FISH for in situ DNA labeling. PRINS is specific, simple, and rapid. We review some applications of PRINS involving primers specific for telomeric, human Alu, and centromeric alpha-satellite sequences. Bicolor labeling, PRINS-FISH, or PRINS-immunofluorescent combinations have been developed to enable investigations in numerous applications.

Ex vivo purging by adenoviral p53 gene therapy does not affect NOD-SCID repopulating activity of human CD34+ cells.

Co-incubation of a replication-deficient, recombinant adenovirus carrying the wild-type p53 gene (rAd-p53) and hematopoietic stem cell (HSC) products from patients with breast cancer can significantly reduce tumor cell contamination. Whereas this approach provides a powerful tumor cell purging strategy, potential detrimental effects on the HSC population have not been investigated. The ability of human HSC to reconstitute hematopoiesis in severe combined immunodeficient (SCID) mice and to undergo secondary transplantation provides the only nonclinical measure of self-renewing, stem cell function. The objective of this study was to investigate whether co-incubation with rAd-p53 compromised the SCID repopulating activity (SRA) of HSC. Granulocyte colony-stimulating factor-mobilized human CD34+ cells were co-cultured with rAd-p53 at our targeted clinical dose, and the ability of these cells to establish multilineage hematopoiesis in sublethally irradiated, nonobese diabetic (NOD)-SCID mice was investigated. The persistence of human cells in the mice was investigated by flow cytometry, granulocyte-macrophage colony-forming unit assay, and polymerase chain reaction of human Alu sequences. Further, limiting dilution analysis provided a quantitative comparison between the SRA of CD34+ cells co-incubated with rAd-p53 and control CD34+ cells (no rAd-p53 co-incubation). We conclude that co-incubation with rAd-p53 has little effect on the SRA of HSC.

The 3′ UTR of Human MnSOD mRNA Hybridizes to a Small Cytoplasmic RNA and Inhibits Gene Expression

Human MnSOD localizes to the mitochondria and plays a key protective role by detoxifying oxygen free radicals. The MnSOD mRNA 3′ UTR contains a 280-bp region (Alu-like element or Alu-E) that shows high homology to human Alu and 7SL sequences. MnSOD 3′ UTR probes hybridize to a specific cytoplasmic RNA species of ∼300 nucleotides. This antisense RNA is most likely 7SL RNA based on its size, ubiquitousness, high levels, and lack of inducibility. Hybridization of this small RNA to the MnSOD 3′ UTR may modulate posttranscriptional MnSOD gene expression. This regulation could occur by several means including inhibition of translation and mRNA destabilization. Regulation at the level of translational initiation does not seem to occur as MnSOD mRNA containing the Alu-E is efficiently bound by ribosomes. To test the role of the MnSOD 3′ UTR, and in particular the Alu-E in gene expression, luciferase reporter gene constructs were made containing various regions of the MnSOD 3′ UTR including the Alu-E. These constructs were transfected into human A549 lung carcinoma cells and luciferase activity was measured. Reporter constructs containing the MnSOD 3′ UTR and the Alu-E repress luciferase activity. Taken together, these results suggest that naturally occurring antisense RNA may bind MnSOD mRNA and repress its expression. These results also suggest that other mRNAs containing Alu elements may be similarly repressed.

Sequence patterns observed in 5' flanking regions of primate Alu elements.

Retrotransposons have generally been known to integrate randomly into host genomes. Jurka, however, showed some consensus sequence patterns at insertion sites of some mammalian retrotransposons and proposed enzymatic involvement that mediates integration. Jurka used about 400 complete human Alu and rodent ID sequences which retain full length with identical flanking sequences at both ends. In our study, more than 25,000 Alu sequences longer than 250 bp were used for comprehensive analysis to identify any consensus sequence(s) preceding the 5' end of Alu elements. "Entropy" at each nucleotide position within 500 bases of the 5' Alu end was computed. Significant drop of entropy was observed between position -20 and -10 of the 5' end of Alu, suggesting the existence of certain consensus sequence(s) in the region. Frequencies of all possible triplets (total of 64) were measured in the same region. Observation that frequencies of triplets "aaa," "taa," and "tta" in the 5' flanking sequences were high is consistent with Jurka. However, frequencies of triplets "att" and "aca" were also significantly high, which are not the primary candidates for nick site in Jurka.

Growth inhibition by a triple ribozyme targeted to repetitive B2 transcripts.

The B2 family represents a group of short repetitive sequences that are found throughout the rodent genome and are analogous to the human Alu sequences. Certain B2 subfamilies are transcribed by RNA polymerase III (pol III), and this transcription is in part controlled by the retinoblastoma protein. In addition to their putative role in retrotranspositional events, these actively transcribed B2 RNAs show a predicted highly stable secondary structure. Although B2 transcripts are normally confined to the nucleus, they demonstrate altered compartmentation after carcinogen treatment, in cancers, and in immortalized and/or transformed cell lines, the significance of which is unclear. Because modulation of B2 transcripts did not seem feasible with an antisense approach, we designed a triple ribozyme (TRz) construct to down-regulate B2 transcripts. The B2-targeted TRz undergoes efficient self-cleavage, resulting in liberation of the internal hammerhead Rz, which we targeted to a single-stranded region of the consensus B2 sequence. The liberated internal targeted Rz was 20 times more active than the corresponding double-G mutant construct that could not undergo self-cleavage, and 5 times more active than the same Rz flanked by nonspecific vector sequences. The B2-targeted TRz was used to develop stable transfectant clones from an SV40-immortalized hepatocyte cell line. These transfectant clones all showed variably reduced growth rates, accompanied by significant reductions in both cytoplasmic and nuclear B2 RNA levels: linear regression analyses showed that their growth rates were directly related to residual cytoplasmic B2 levels. Reverse-transcription polymerase chain reaction (RT-PCR) analyses documented efficient self-liberation of the internal targeted Rz in vivo, and showed that the relative cytoplasmic expression levels generally paralleled the magnitude of the decrease in B2 transcripts. The RT-PCR analyses further demonstrated that up to 20% of the Rz was located in the nucleus, which presumably reflects competition between autocatalytic processing and nucleocytoplasmic transport of the initial TRz transcript.

Requirement for Specific Proteases in Cancer Cell Intravasation as Revealed by a Novel Semiquantitative PCR-Based Assay

Proteases are crucial for cancer metastasis, but due to lack of assays, their role in intravasation has not yet been tested. We have developed a human Alu sequence PCR-based assay to quantitate intravasated cells in an in vivo model. We demonstrated that metalloproteinases (MMPs), and most likely MMP-9, are required for intravasation by showing that marimastat, an inhibitor of MMPs, reduced intravasation by more than 90%, and that only tumor cell lines expressing MMP-9 intravasated. Cells with low surface urokinase plasminogen activator (uPA) and uPA receptor (uPAR) were also incapable of intravasation, despite the presence of high levels of MMP-9. We concluded that breaching of the vascular wall is a rate-limiting step for intravasation, and consequently for metastasis, and that cooperation between uPA/uPAR and MMP-9 is required to complete this step.

Inter-Alu PCR like genomic profiling in banana

Alu sequences are a major repetitive element of the primate genome. To date Alu sequences have seldom been reported in plant genomes. We report here an inter-Alu PCR like genomic profiling in banana using a single primer designed from a human Alu sequence. The different Musa species involved in the complex genome of banana cultivars can thus be discriminated. The use of this technique for monitoring germplasm or genotyping cultivars is discussed.

Genomic organization of DHR38 gene in Drosophila: presence of Alu-like repeat in a translated exon and expression during embryonic development.

The recombinant lambda clone 4-2 containing the genomic region of the Drosophila hormone receptor 38 (DHR 38) gene, homologous to mammalian neuronal growth factor I-B (NGFI-B), was isolated by radioactive labelled oligonucleotide hybridization. The nucleotide sequence of the genomic clone revealed three exons that encode the functional domains of the protein. The N-terminal exon1 had no homology at the amino acid level with NGFI-B, the mammalian homologue. A glutamine-rich region, probably involved in transcriptional activation, was observed at the C-terminal part of this exon. A similar motif is also present upstream in another reading frame of the same strand. Both motifs are preceded by a repetitive non-anucleotide sequence containing an AluI site, resembling a duplicated human Alu-sequence. A monomeric version of this sequence, coding similarly for an oligoglutamine peptide, occurs at a surprisingly high frequency in other regulatory genes in Drosophila. In contrast to mammalian Alu sequences, this sequence is found almost exclusively in the coding regions of Drosophila genes, but not in the non-coding parts of the genes. The DNA-binding domain with two zinc-fingers, and at least part of the ligand-binding peptide, is coded by the largest middle exon2 in DHR38 and exhibits up to 100% homology in short peptide motifs to its mammalian counterpart, where these domains are split into exons 3, 4, 5, and 6. However, the length, information content, stop codon, and splice site are conserved in the last exons in both fly and rat. In situ hybridization to 0-24 h wholemount embryos showed strong expression of DHR38 in neurogenic regions and in the intestinal tract during embryogenesis, suggesting a spatial and temporal control of transcription, partially analogous to the central nervous system-specific expression of NGFI-B in mammals.

Estimating the age of the common ancestor of a sample of DNA sequences.

We present a simple Monte Carlo method for estimating the age of the most recent common ancestor (MRCA) of a sample of DNA sequences. We show that Templeton's (1993) estimator of the age of the MRCA based on the maximum number of nucleotide differences between two sequences in a sample is inaccurate, and we demonstrate the new method by reanalyzing a sample of DNA sequences from human Y chromosomes and a sample of human Alu sequences.

A Population Genetic Study of the Evolution of SINEs. II. Sequence Evolution Under the Master Copy Model

A transient population genetic model of SINE (short interspersed repetitive element) evolution assuming the master copy model is theoretically investigated. Means and variances of consensus frequency of nucleotides, nucleotide homozygosity, and the number of shared differences that are considered to have caused by mutations occurring in the master copy lineages are computed. All quantities investigated are shown to be monotone functions of the duration of the expansion period. Thus, they can be used to estimate the expansion period although their sampling variances are generally large. Using the theoretical results, the Sb subfamily of human Alu sequences is analyzed. First, the expansion period is estimated from the observed mean and variance of homozygosity. The expansion period is shown to be short compared to the time since the end of the expansion of the subfamily. However, the observed number of the shared differences is more than twice that expected under the master copy model with the estimated expansion period. Alternative models including that with multiple master copy loci to explain this observation are discussed.

SINE sequences detect DNA fingerprints in salmonid fishes

DNA probes homologous to two previously described salmonid short interspersed nuclear elements (SINEs) detected DNA fingerprint patterns in 14 species of salmonid fishes. The probes showed more homology to some species than to others and little homology to three nonsalmonid fishes. The DNA fingerprint patterns derived from the SINE probes are individual-specific and inherited in a Mendelian manner. Probes derived from different regions of the same SINE detect only partially overlapping banding patterns, reflecting a more complex SINE structure than has been previously reported. Like the human Alu sequence, the SINEs found in salmonids could provide useful genetic markers and primer sites for PCR-based techniques. These elements may be more desirable for some applications than traditional DNA fingerprinting probes that detect tandemly repeated arrays.

Specific cloning of human DNA as yeast artificial chromosomes by transformation-associated recombination.

DNA molecules undergoing transformation into yeast are highly recombinogenic, even when diverged. We reasoned that transformation-associated recombination (TAR) could be employed to clone large DNAs containing repeat sequences, thereby eliminating the need for in vitro enzymatic reactions such as restriction and ligation and reducing the amount of DNA handling. Gently isolated human DNA was transformed directly into yeast spheroplasts along with two genetically marked (M1 and M2) linearized vectors that contained a human Alu sequence at one end and a telomere sequence at the other end (Alu-CEN-M1-TEL and Alu-M2-TEL). Nearly all the M1-selected transformants had yeast artificial chromosomes (YACs) containing human DNA inserts that varied in size from 70 kb to > 600 kb. Approximately half of these had also acquired the unselected M2 marker. The mitotic segregational stability of YACs generated from one (M1) or two (M1 and M2) vector(s) was comparable, suggesting de novo generation of telomeric ends. Since no YACs were isolated when rodent DNAs or a vector lacking an Alu sequence was used, the YACs were most likely the consequence of TAR between the repeat elements on the vector(s) and the human DNA. Using the BLUR13 Alu-containing vector, we demonstrated that human DNA could be efficiently cloned from mouse cells that contained a single human chromosome 16. The distribution of cloned DNAs on chromosome 16 was determined by fluorescence in situ hybridization. We propose that TAR cloning can provide an efficient means for generating YACs from specific chromosomes and subchromosome fragments and that TAR cloning may be useful for isolating families of genes and specific genes from total genome DNA.

Computer Analyses of Site-Specific Variabilities in Human Alu Sequences

Computer Analyses of Site-Specific Variabilities in Human Alu Sequences

Transcriptional silencing of human Alu sequences and inhibition of protein binding in the box B regulatory elements by 5′-CG-3′ methylation

In earlier work, we demonstrated that 5′-CG-3′ methylation inhibits the transcriptional activity of human Alu elements associated with the α 1-globin and the angiogenin genes in a cell-free transcription system from HeLa nuclear extracts. These studies have been extended to different Alu sequences and to investigations on the mechanism involved in transcriptional silencing by methylation. By comparing the results of DNase I and dimethyl sulfate (DMS) in vitro footprinting on a consensus sequence in the RNA polymerase III promoter control B region between the unmethylated and the 5′-CG-3′ methylated B box, evidence has been adduced for effects of 5′-CG-3′ methylation on the interaction of specific nuclear proteins with DNA sequences in the B control region of the Alu elements. These results are consistent with the interpretation that the 5′-CG-3′ methylation interferes with the binding of proteins that are essential for the function of the B control region in these RNA polymerase III-transcribed elements, and that promoter methylation thus inhibits transcription.

Detection and cloning of potent transforming gene(s) from chewing tobacco-related human oral carcinomas

High molecular weight DNA isolated from 14 primary tumour tissues of human oral carcinoma patients was analysed for transforming activity by NIH3T3 co-transfection assay using pSV2neo gene as a selectable marker, followed by nude mouse tumorigenicity assay. Ten of the patient tumour tissues demonstrated molecular lesions in myc, ras or/and EGF-R genes, whereas 4 patients did not show tumour associated aberrations in these oncogenes. The G418-resistant transfected cells from 12 of 14 individual patients demonstrated transforming potential by colony formation in soft agar and tumour induction in nude mice within 25-80 days. DNAs from the transfected cells, consequent nude mice tumours and corresponding cell lines, contained human Alu sequences. Southern blot hybridisation with ras, myc, EGF-R oncogenes demonstrated the presence of human H-ras oncogene in one of the 12 sets of nude mice tumours. In contrast, DNA from the other 11 sets of nude mice tumours indicated absence of c-myc, N-myc, L-myc, H-ras, K-ras, N-ras and EGF-R genes on Southern analysis. Further, DNAs from five first cycle tumorigenic transformants were subjected to a second cycle of transfection, and induced tumours in nude mice with a shorter latency period of 21-50 days. The secondary transformants contained discrete human Alu sequences; however, the DNA did not hybridise with myc/ras/EGF-R probes. A genomic library was constructed from a second cycle nude mice tumour, using EMBL-3 as the vector. Four human Alu sequence positive clones were isolated on screening 2 x 10(5) plaques, and one of the recombinant clones subjected to fine restriction mapping using 16 restriction enzymes. The lack of association of the nude mice tumour DNA with myc/ras/EGF-R showing aberrations in the primary human tumour, implies activation of an alternative potent transforming gene(s) in the chewing tobacco-related oral carcinomas in India.

Evidence that most human Alu sequences were inserted in a process that ceased about 30 million years ago.

The primate Alu interspersed repeats can be subdivided into classes on the basis of shared nucleotides at a set of diagnostic positions. Each of the classes of Alu sequences is apparently the result of past retrotransposition of transcripts of highly conserved class-specific source genes that differed from each other at the diagnostic positions. The nucleotides at the majority of positions are identical among the source genes and therefore were identical among all of the Alu sequences at the time of their insertion. These CONSBI (conserved before insertion) positions are useful because the changes that have occurred after insertion are recognizable and the divergence resulting from nucleotide substitutions, insertions, and deletions is informative. The divergence of Alu sequences at the CONSBI positions is a measure of the time since a class was inserted. The greatest majority of Alu sequences are in one class (identified as class II), and it is particularly suitable for such examination, since nearly full-length sequences are now known for nearly a thousand members of this class. The average divergence of class II members indicates that the class has an average age of about 40 million years. The distribution in divergence of class II accurately fits a sum of two Poisson distributions. The implication is that class II Alu sequences were derived from two massive past events of insertion of many Alu sequences. In this model the younger subset of class II sequences (corresponding to about 300,000 copies in the genome) has an average divergence of 5% at CONSBI positions. The older set of class II sequences (corresponding to about 150,000 genomic copies) has a 9% average divergence. Based on the drift rate of primate DNA sequences, the events of insertion probably occurred 30-50 million years ago. The goodness of the fit to the Poisson distribution indicates that no significant number of members of class II have been inserted since 30 million years ago.

A human Alu RNA-binding protein whose expression is associated with accumulation of small cytoplasmic Alu RNA.

Human Alu sequences are short interspersed DNA elements which have been greatly amplified by retrotransposition. Although initially derived from the 7SL RNA component of signal recognition particle (SRP), the Alu sequence has evolved into a dominant transposon while retaining a specific secondary structure found in 7SL RNA. We previously characterized a set of Alu sequences which are expressed as small cytoplasmic RNAs and isolated a protein that binds to these transcripts. Here we report that biochemical purification of this protein revealed it as the human homolog of the SRP 14 polypeptide which binds the Alu-homologous region of 7SL RNA. The human cDNA predicts an alanine-rich C-terminal tail translated from a trinucleotide repeat not found in the rodent homolog, which accounts for why the human protein-RNA complex migrates more slowly than its rodent counterpart in RNA mobility shift assays. The human Alu RNA-binding protein (RBP) is expressed after transfection of this cDNA into mouse cells. Expression of human RBP in rodent x human somatic cell hybrids is associated with substantial increase in endogenous small cytoplasmic Alu and scB1 transcripts but not other small RNAs. These studies provide evidence that this RBP associates with Alu transcripts in vivo and affects their metabolism and suggests a role for Alu transcripts in translation in an SRP-like manner. Analysis of hybrid lines indicated that the Alu RBP gene maps to human chromosome 15q22, which was confirmed by Southern blotting. The possibility that the primate-specific structure of this protein may have contributed to Alu evolution is considered.

A human Alu RNA-binding protein whose expression is associated with accumulation of small cytoplasmic Alu RNA.

Human Alu sequences are short interspersed DNA elements which have been greatly amplified by retrotransposition. Although initially derived from the 7SL RNA component of signal recognition particle (SRP), the Alu sequence has evolved into a dominant transposon while retaining a specific secondary structure found in 7SL RNA. We previously characterized a set of Alu sequences which are expressed as small cytoplasmic RNAs and isolated a protein that binds to these transcripts. Here we report that biochemical purification of this protein revealed it as the human homolog of the SRP 14 polypeptide which binds the Alu-homologous region of 7SL RNA. The human cDNA predicts an alanine-rich C-terminal tail translated from a trinucleotide repeat not found in the rodent homolog, which accounts for why the human protein-RNA complex migrates more slowly than its rodent counterpart in RNA mobility shift assays. The human Alu RNA-binding protein (RBP) is expressed after transfection of this cDNA into mouse cells. Expression of human RBP in rodent x human somatic cell hybrids is associated with substantial increase in endogenous small cytoplasmic Alu and scB1 transcripts but not other small RNAs. These studies provide evidence that this RBP associates with Alu transcripts in vivo and affects their metabolism and suggests a role for Alu transcripts in translation in an SRP-like manner. Analysis of hybrid lines indicated that the Alu RBP gene maps to human chromosome 15q22, which was confirmed by Southern blotting. The possibility that the primate-specific structure of this protein may have contributed to Alu evolution is considered.