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This review presents some properties of interspersed repeats, particularly human and mouse repeats, and shows how these have been utilized in long-range genome mapping. The link between the distribution of such repeats and their relationship with genome organization is discussed.
Cot and chemical analysis show that the haploid genome size of Thermobia domestica is 3-4x10(9) nucleotide pairs. Of this DNA 33% is single copy sequences and 67% is repetitive sequences. The repetitive sequences are predominantly 300 nucleotides in length and are interspersed among the single copy sequences in a short period interspersion pattern similar to that observed in Xenopus and many other higher eucaryotes. The DNA sequence organization observed in Thermobia is compared with that of other more highly evolved insects.
Subtelomeric regions have been a target of structural and functional studies of human chromosomes. Markers having a defined structure are especially useful to such studies. Here, we report 93 bp tandem repeat sequences found in the subtelomeric region of human chromosome 21q. They were also detected in the telomeric region of several other chromosomes. Interestingly, the repeat was also found in the 2q13 region which is known to be a position of chromosomal fusion, a major difference between the human and chimpanzee karyotypes. To the best of our knowledge, this repetitive sequence is a new member of human subtelomeric interspersed repeats.
Mycobacterial interspersed repetitive units (MIRUs), a novel class of repeated sequences, were identified within the intercistronic region of an operon coding for a mycobacterial two-component system, named senX3-regX3. Southern blot analysis and homology searches revealed the presence of several homologous sequences in intergenic regions dispersed throughout the genomes of Mycobacterium bovis BCG, Mycobacterium tuberculosis and Mycobacterium leprae. These could be grouped into three major families, containing elements of 77-101 bp, 46-53 bp and 58-101 bp. Based on the available mycobacterial sequences, the total number of MIRUs is estimated to be about 40-50 per genome. Similar to previously identified small repetitive sequences, the MIRUs of the two-component operon are transcribed on a polycistronic mRNA. Unlike previously identified small repetitive sequences, however, MIRUs do not contain dyad symmetries, comprise small open reading frames (ORFs) whose extremities overlap those of the contiguous ORFs and are oriented in the same translational direction as those of the adjacent genes. Analyses of the sequences at the insertion sites suggest that MIRUs disseminate by transposition into DTGA sites involved in translational coupling in polycistronic operons.
Lyon has proposed that long interspersed nuclear element 1 (LINE-1 or L1) repeats may be mediators for the spread of X chromosome inactivation. Cells from ICF patients who are deficient in one of the DNA methyltransferases, DNMT3B, provide an opportunity to explore and refine this hypothesis. Southern blot and bisulfite methylation analyses indicate that, in normal somatic cells, X-linked L1s are hypermethylated on both the active and inactive X chromosomes. In contrast, ICF syndrome cells with DNMT3B mutations have L1s that are hypomethylated on the inactive X, but not on the active X or autosomes. The DNMT3B methyltransferase, therefore, is required for methylation of L1 CpG islands on the inactive X, whereas methylation of the corresponding L1 loci on the active X, as well as most autosomal L1s, is accomplished by another DNA methyltransferase. This unique phenomenon of identical allelic modifications by different enzymes has not been previously observed. Apart from CpG island methylation, the ICF inactive X is basically normal in that it forms a Barr body, is associated with XIST RNA, mostly replicates late, and its X-inactivated genes are mostly silent. Because the unmethylated state of the ICF inactive X L1s probably reflects their methylation status at the time of X inactivation, these data suggest that unmethylated L1 elements, but not methylated L1s, may have a role in the spreading of X chromosome inactivation.
Manometric criteria for diffuse esophageal spasm have recently been restated. In this study, a cohort of 358 subjects was evaluated in a gastrointestinal motility laboratory for dysphagia and/or chest pain. Applying the recently proposed criteria of Richter and Castell, 18 subjects (5%) were diagnosed as having DES. Dysphagia was the major complaint (89%), while 44% of patients complained of chest pain and 33% of both symptoms. All patients shared more than 30% simultaneous contractions after wet swallows interspersed with normal peristaltic sequences. Associated manometric findings were repetitive (greater than 3 peaks) contractions (67%), high-amplitude contractions (33%), spontaneous activity (22%), prolonged duration (11%), and lower esophageal sphincter abnormalities (5%). Radiology disclosed significant abnormalities in only 27% of DES patients.
The chromosomal gene expression profiles established by the Human Transcriptome Map (HTM) revealed a clustering of highly expressed genes in about 30 domains, called ridges. To physically characterize ridges, we constructed a new HTM based on the draft human genome sequence (HTMseq). Expression of 25,003 genes can be analyzed online in a multitude of tissues (http://bioinfo.amc.uva.nl/HTMseq). Ridges are found to be very gene-dense domains with a high GC content, a high SINE repeat density, and a low LINE repeat density. Genes in ridges have significantly shorter introns than genes outside of ridges. The HTMseq also identifies a significant clustering of weakly expressed genes in domains with fully opposite characteristics (antiridges). Both types of domains are open to tissue-specific expression regulation, but the maximal expression levels in ridges are considerably higher than in antiridges. Ridges are therefore an integral part of a higher order structure in the genome related to transcriptional regulation.
DNA extracted from Chironomus thummi larvae was studied by isopycnic centrifugation in CsCl, thermal denaturation and DNA-DNA reassociation techniques. The mean G+C content of the C. thummi DNA is 28-29% as indicated both by centrifugation in CsCl and thermal denaturation. According to optical reassociation analysis of total DNA and of isolated DNA fractions the C. thummi genome is composed of at least four components. About 80% of the DNA is classified as unique with a kinetic complexity of nearly 7 X 10(10) daltons. 6-8% intermediate DNA exhibits a kinetic complexity slightly above 10(8) daltons with a mean repetition frequency of 35. 11-13% fast-reassociating DNA has a kinetic complexity slightly above 10(6) daltons with a mean repetition frequency of 6000. 3-5% of the DNA cannot be properly studied by the optical reassociation technique and probably contains inverted repeats. The thermal denaturation behaviour of isolated DNA fractions indicated that most of the repetitive sequences in the C. thummi genome are tightly interspersed.
Short interspersed repetitive elements (SINEs) are a type of retroposon, being members of a class of informational molecules that are amplified via cDNA intermediates and flow back into the host genome. In contrast to retroviruses and retrotransposons, SINEs do not encode the enzymes required for their amplification, such as reverse transcriptases, so they are presumed to borrow these enzymes from other sources. In the present study, we isolated a family of long interspersed repetitive elements (LINEs) from the turtle genome. The sequence of this family was found to be very similar to those of the avian CR1 family. To our surprise, the sequence at the 3' end of the LINE in the turtle genome was nearly identical to that of a family of tortoise SINEs. Since CR1-like LINEs are widespread in birds and in many other reptiles, including the turtle, and since the tortoise SINEs are only found in vertical-necked turtles, it seems possible that the sequence at the 3' end of the tortoise SINEs might have been generated by recombination with the CR1-like LINE in a common ancestor of vertical-necked turtles, after the divergence of side-necked turtles. We extended our observations to show that the 3'-end sequences of families of several tRNA-derived SINEs, such as the salmonid HpaI family, the tobacco TS family, and the salmon SmaI family, might have originated from the respective LINEs. Since it appears reasonable that the recognition sites of LINEs for reverse transcriptase are located within their 3'-end sequences, these results provide the basis for a general scheme for the mechanism by which SINEs might acquire retropositional activity. We propose here that tRNA-derived SINEs might have been generated by a recombination event in which a strong-stop DNA with a primer tRNA, which is an intermediate in the replication of certain retroviruses and long terminal repeat retrotransposons, was directly integrated at the 3' end of a LINE.
Xlsirts are a family of interspersed repeat RNAs from Xenopus laevis that contain from 3 to 13 repeat units (each 79 to 81 nucleotides long) flanked by unique sequences. They are homologous to the mammalian Xist gene that is involved in X chromosome inactivation. Xlsirt RNA appears first in the mitochondrial cloud (Balbiani body) in stage 2 oocytes and is then translocated as island-like structures to the vegetal cortex at early stage 3 coincident with the localization of the germ plasm. Exogenous Xlsirt RNA injected into oocytes translocates to the location of the endogenous RNA at that particular stage. The Xlsirt RNA repeat sequences are required for translocation and can cause the translocation of heterologous unique RNAs to the vegetal cortex.
Alu repeats are interspersed repetitive DNA elements specific to primates that are present in 500,000 to 1 million copies. We show here that an Alu sequence encodes functional binding sites for retinoic acid receptors, which are members of the nuclear receptor family of transcription factors. The consensus sequences for the evolutionarily recent Alu subclasses contain three hexamer half sites, related to the consensus AGGTCA, arranged as direct repeats with a spacing of 2 bp, which is consistent with the binding specificities of retinoic acid receptors. An analysis was made of the DNA binding and transactivation potential of these sites from an Alu sequence that has been previously implicated in the regulation of the keratin K18 gene. These Alu double half sites are shown to bind bacterially synthesized retinoic acid receptors as assayed by electrophoretic mobility shift assays. These sites are further shown to function as a retinoic acid response element in transiently transfected CV-1 cells, increasing transcription of a reporter gene by a factor of approximately 35-fold. This transactivation requires cotransfection with vectors expressing retinoic acid receptors, as well as the presence of all-trans-retinoic acid, which is consistent with the known function of retinoic acid receptors as ligand-inducible transcription factors. The random insertion of potentially thousands of Alu repeats containing retinoic acid response elements throughout the primate genome is likely to have altered the expression of numerous genes, thereby contributing to evolutionary potential.
We report a collection of 53 prototypic sequences representing known families of repetitive elements from the human genome. The prototypic sequences are either consensus sequences or selected examples of repetitive sequences. The collection includes: prototypes for high and medium reiteration frequency interspersed repeats, long terminal repeats of endogenous retroviruses, alphoid repeats, telomere-associated repeats, and some miscellaneous repeats. The collection is annotated and available electronically.
The reassociation kinetics of pea (Pisum sativum L.) DNA fragments (300 nucleotides) were measured with hydroxylapatite. The most slowly reassociating fragments do so with a rate constant of 2 X 10(-4) L mol-1s-1, as determined from experiments with total DNA as well as with a tracer enriched for slowly renaturing sequences. This rate is about 1000 times slower than that observed for Escherichia coli DNA included as an internal kinetic standard, indicating a kinetic complexity of 4.5 X 10(9) nucleotide pairs or 4.6 pg of DNA per haploid nucleus. This estimate is in good agreement with previous chemical and cytophotometric measurements. The majority (85%) of the 300 nucleotide fragments contain repetitive sequences. While the reassociation of repetitive DNA could be modeled with two theoretical second-order components, the data did not specify a unique solution. The reassociation kinetics of isolated high- and low-frequency fractions indicate that repetitive sequence families in pea DNA probably cover a broad range of frequencies ranging from 100 to 10 000 or more copies per haploid genome. Single-copy sequences account for about 30% of the DNA, but because of extensive interpersion of repetitive sequences only about 15% of 300 nucleotide fragments reassociate with single-copy kinetics. From studies of hydroxylapatite binding as a function of fragment length, we conclude that the major class of single-copy sequences has a modal length of about 300 nucleotides. Long tracer reassociation kinetics indicate that sequences with an apparent repetition frequency of about 10 000 copies are interspersed at intervals of less than 1300 nucleotides throughout 75% of the genome. At a detection limit of about 3%, we find no single-copy sequences longer than 1000 nucleotides.
Genes that play a role in the senescent arrest of cellular replication are likely to be overexpressed in human diploid fibroblasts (HDF) derived from subjects with Werner syndrome (WS) because these cells have a severely curtailed replicative life span. To identify some of these genes, a cDNA library was constructed from WS HDF after they had been serum depleted and repleted (5 days in medium containing 1% serum followed by 24 h in medium containing 20% serum). Differential screening of 7,500 colonies revealed 102 clones that hybridized preferentially with [32P]cDNA derived from RNA of WS cells compared with [32P]cDNA derived from normal HDF. Cross-hybridization and partial DNA sequence determination identified 18 independent gene sequences, 9 of them known and 9 unknown. The known genes included alpha 1(I) procollagen, alpha 2(I) procollagen, fibronectin, ferritin heavy chain, insulinlike growth factor-binding protein-3 (IGFBP-3), osteonectin, human tissue plasminogen activator inhibitor type I, thrombospondin, and alpha B-crystallin. The nine unknown clones included two novel gene sequences and seven additional sequences that contained both novel segments and the Alu class of repetitive short interspersed nuclear elements; five of these seven Alu+ clones also contained the long interpersed nuclear element I (KpnI) family of repetitive elements. Northern (RNA) analysis, using the 18 sequences as probes, showed higher levels of these mRNAs in WS HDF than in normal HDF. Five selected mRNAs studied in greater detail [alpha 1(I) procollagen, fibronectin, insulinlike growth factor-binding protein-3, WS3-10, and WS9-14] showed higher mRNA levels in both WS and late-passage normal HDF than in early-passage normal HDF at various intervals following serum depletion/repletion and after subculture and growth from sparse to high-density confluent arrest. These results indicate that senescence of both WS and normal HDF is accompanied by overexpression of similar sets of diverse genes which may play a role in the senescent arrest of cellular replication and in the genesis of WS, normal biological aging, and attendant diseases.
Palindromic units (PU or REP) were defined as 40-nucleotide DNA sequences which are highly repeated in the genome of several members of the Enterobacteriaceae. They were shown to be a constituent of the bacterial interspersed mosaic element (BIME), in which they are associated with other repetitive sequences. We report here that Escherichia coli PU sequences contain three motifs (Y, Z1 and Z2), leading to the definition of two BIME families. The BIME-1 family, highly conserved over 145 nucleotides, contains two PUs (motifs Y and Z1). The BIME-2 family contains a variable number of PUs (motifs Y and Z2). We present evidence, using band shift experiments, that each PU motif binds DNA gyrase with a different affinity. This suggests that the two families are functionally distinct.
Experiments are described that characterize the organization of DNA sequences in the water mold Achlya bisexualis. These experiments demonstrate that repetitive and single copy sequences in the Achlya genome are arranged in a long-period interspersion pattern. Estimates of the spacing intervals between repetitive and single copy DNA indicate, however, that the interspersion pattern in Achlya is longer than has been previously reported in other eukaryotes. These data and measurements of structural gene expression in Achlya [Timberlake, W.E., Shumard, D. S. & Goldberg, R. B. (1977) Cell 10, 623-632] make it difficult to propose a regulatory function for repeated DNA in this eukaryote.
The size and arrangement of repetitive and inverted repeat (foldback) sequences in rat DNA were studied by visualization of hybrid and heteroduplex structures in the electron microscope. The self-reassociation of repetitive sequence-bearing DNA strands often results in the formation of four-ended "H" structures, whose duplex regions equal the repetitive sequence length and can be measured in the electron microscope. In this way, it was determined that the average size of the class of numerous short repetitive sequences is 0.40 +/- 0.15 kbp. Heteroduplex structures were prepared between long whole DNA single strands and short repeat-sequence-bearing strands. The analysis of these structures confirms that the size of the repetitive sequences in 0.4 kbp on average. Length measurements between adjacent duplexes show that the average spacing between two interspersed repeats is at least 1.5-1.8 kbp. By examining 29.4-kbp single strands after brief renaturation, the size and distribution of foldback sequences were determined. There are 1.9 X 10(5) foldback apirs per rat genome, spaced an average of 9.7 kbp apart according to our measurement. Repetitive, inverted repeat and unique sequences are interspersed with each other in at least half the genome.
We have isolated and characterized from rice three repetitive DNA sequences, Os48, Osc-567, and OsG3-430. Our results indicate that these repetitive sequences are highly transcribed, and transcripts complementary to both strands of the Os48 family of sequences account for up to 3% of the total cellular RNA. Pulsed-field gel electrophoresis, restriction mapping, and DNA sequence analyses have revealed a complex pattern of structural organization of the three families of repetitive sequences. Os48 and Osc-567 are organized in long tandem arrays, whereas copies of the OsG3-340 sequence are interspersed with other sequences including arrays of the Os48 and Osc567 families. Interestingly, the three families of repetitive sequences are closely linked not only to each other, but also to telomeric sequences of rice, suggesting that transcription of these repetitive sequences may occur in regions very close to telomeres in rice.