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PubMed2005

Plant viral intergenic DNA sequence repeats with transcription enhancing activity.

BACKGROUND: The geminivirus and nanovirus families of DNA plant viruses have proved to be a fertile source of viral genomic sequences, clearly demonstrated by the large number of sequence entries within public DNA sequence databases. Due to considerable conservation in genome organization, these viruses contain easily identifiable intergenic regions that have been found to contain multiple DNA sequence elements important to viral replication and gene regulation. As a first step in a broad screen of geminivirus and nanovirus intergenic sequences for DNA segments important in controlling viral gene expression, we have 'mined' a large set of viral intergenic regions for transcriptional enhancers. Viral sequences that are found to act as enhancers of transcription in plants are likely to contribute to viral gene activity during infection. RESULTS: DNA sequences from the intergenic regions of 29 geminiviruses or nanoviruses were scanned for repeated sequence elements to be tested for transcription enhancing activity. 105 elements were identified and placed immediately upstream from a minimal plant-functional promoter fused to an intron-containing luciferase reporter gene. Transient luciferase activity was measured within Agrobacteria-infused Nicotiana tobacum leaf tissue. Of the 105 elements tested, 14 were found to reproducibly elevate reporter gene activity (>25% increase over that from the minimal promoter-reporter construct, p < 0.05), while 91 elements failed to increase luciferase activity. A previously described "conserved late element" (CLE) was identified within tested repeats from 5 different viral species was found to have intrinsic enhancer activity in the absence of viral gene products. The remaining 9 active elements have not been previously demonstrated to act as functional promoter components. CONCLUSION: Biological significance for the active DNA elements identified is supported by repeated isolation of a previously defined viral element (CLE), and the finding that two of three viral enhancer elements examined were markedly enriched within both geminivirus sequences and within Arabidopsis promoter regions. These data provide a useful starting point for virologists interested in undertaking more detailed analysis of geminiviral promoter function.

Arabidopsis↗

PubMed1989

Host species-specific conservation of a family of repeated DNA sequences in the genome of a fungal plant pathogen.

We have identified a family of dispersed repetitive DNA sequences in the genome of Magnaporthe grisea, the fungus that causes rice blast disease. We have named this family of DNA sequences "MGR" for M. grisea repeat. Analysis of five MGR clones demonstrates that MGR sequences are highly polymorphic. The segregation of MGR sequences in genetic crosses and hybridization of MGR probes to separated, chromosome-size DNA molecules of M. grisea shows that this family of sequences is distributed among the M. grisea chromosomes. MGR sequences also hybridize to discrete poly(A)+ RNAs. Southern blot analysis using a MGR probe can distinguish rice pathogens from various sources. However, MGR sequences are not highly conserved in the genomes of M. grisea field isolates that do not infect rice. These results suggest that host selection for a specific pathogen genotype has occurred during the breeding and cultivation of rice.

Ascomycota↗

PubMed1991

Use of short sequence repeat DNA polymorphisms after PCR amplification to detect the parental origin of the additional chromosome 21 in Down syndrome.

The origin of nondisjunction in trisomy 21 has so far been studied using cytogenetic heteromorphisms and DNA polymorphisms using Southern blot analysis. Short sequence repeats have recently been described as an abundant class of DNA polymorphisms in the human genome, which can be typed using the polymerase chain reaction (PCR) amplification. We describe the usage of such markers on chromosome 21 in the study of parental origin of the additional chromosome 21 in 87 cases of Down syndrome. The polymorphisms studied were (a) two (GT)n repeats and a poly(A) tract of an Alu sequence within the HMG14 gene and (b) a (GT)n repeat of locus D21S156. The parental origin was determined in 68 cases by studying the segregation of polymorphic alleles in the nuclear families (either by scoring three different alleles in the proband or by dosage comparison of two different alleles in the proband). Our results demonstrate the usefulness of highly informative PCR markers for the study of nondisjunction in Down syndrome.

Alleles↗

PubMed1986

Elucidation of the B-genome donor to Triticum turgidum by unique- and repeated-sequence DNA hybridizations.

In vitro DNA:DNA hybridizations and hydroxyapatite thermal-elution chromatography were employed to identify the diploid Triticum species ancestral to the B genome of T. turgidum. Unique and repeated sequences from the various Triticum species were separated by hybridization and thermal elution on hydroxyapatite. Unique- and repeated-sequence fractions of labeled T. turgidum var. durum DNA were hybridized to the corresponding fractions of unlabeled DNAs of T. searsii, T. speltoides, T. longissimum, T. sharonensis, and T. bicorne. Thermal stability profiles were constructed to evaluate base-sequence complementarity between T. turgidum var. durum and the diploid Triticum species. The heteroduplex thermal stabilities indicated that, of the five species examined, T. searsii was the most closely related to the B genome of T. turgidum var. durum. The thermal stability profiles further indicated that the repeated DNA fractions from the Triticum species are more similar than the unique-sequence fractions. This indicates that all of the Triticum species are very closely related and, in all probability, have diverged from a single progenitor species.

DNA↗

PubMed1989

Linkage in human heterochromatin between highly divergent Sau3A repeats and a new family of repeated DNA sequences (HaeIII family).

The hybridization of human DNA with three non-cross-hybridizing monomers (68 bp in length) of the heterochromatic Sau3A family of DNA repeats, indicates the coexistence within a Sau3A-positive genomic block of divergent Sau3A units as well as of unrelated sequences. To gain some insight into the structure of these human heterochromatic DNA regions, three previously cloned Sau3A-positive genomic fragments (with a total length of approximately 1900 base-pairs (bp] were sequenced. The analysis of the sequences showed the presence of clustered Sau3A units with different degrees of divergence and of two DNA regions of approximately 100 bp and 291 bp in length, unrelated to the family of repeats. A consensus sequence derived from the 24 identified Sau3A monomers presents, among highly variable regions, two less variant regions of 8 bp and 10 bp in length, respectively. The Sau3A-unrelated DNA fragment 291 bp in length, used as a probe on genomic DNA digested with a series of restriction enzymes, defines a "new" family of DNA repeats possessing periodicities for HaeIII (HaeIII family). Sau3A and HaeIII repeats display a high degree of linkage in a collection of Sau3A-positive genomic recombinant phages.

Base Sequence↗

PubMed2006

Tandemly repeated DNA sequence instabilities induced by two promoters of morphological transformation in vitro: a short-term response to non-mutagenic agents in C3H/10T1/2 cells.

The ability of tumour promoters to alter DNA stability within regions that contain tandemly repeated sequences (TRSs), was studied in a cell culture model of multi-stage carcinogenesis. Non-cytotoxic concentrations of TPA (12-O-tetradecanoyl-phorbol-13-acetate) and xanthine oxidase with xanthine substrate, sufficient to promote morphological transformation in C3H/10T1/2 cultures, were tested for their effects on mutation frequencies in TRSs by a DNA fingerprinting approach. Specifically, restriction digests of genomic DNA samples from randomly selected, non-transformed clones, isolated from cultures after several days exposure to promoters, were visualized by Southern hybridizations with the multi-locus pentamer repeat sequence probe, Ms6-Hm (Pc-1). Basal and promoter-induced frequencies of sub-clone TRS fingerprint polymorphisms were estimated in five cell populations: an uncloned stock culture, three populations established from normal-appearing sub-clones, and one clonal population established from a 3-methylcholanthrene (MCA)-transformed focus. Basal variant fingerprint frequencies spanned a range from 0.0 to 0.43% mutants/band among cells from the four untransformed populations. Both TPA and xanthine oxidase treatments significantly increased recorded mutation frequencies, 2.3- and 2.7-fold, respectively, using combined data from the progenitor population and three untransformed clones. The untreated MCA-transformed clonal population appeared to contain a single, pre-existing mutant restriction fragment, but additional mutations were induced thereafter, in response to the promoting treatments. Taken together, the measured increases in mutations were highly significant and suggest that promoters of cell transformation in the C3H/10T1/2 cell line might induce a genome-wide instability targeted to regions containing Ms6-Hm sequence motifs.

Animals↗

PubMed1987

The 20 bp, directly repeated DNA sequence of broad host range plasmid R1162 exerts incompatibility in vivo and inhibits R1162 DNA replication in vitro.

The broad host range plasmid R1162 contains a directly repeated, 20 bp DNA sequence in the region of the plasmid required in cis for replication and maintenance. This sequence has been chemically synthesized and cloned, and shown to be sufficient for expression of plasmid incompatibility. The sequence also inhibits replication of R1162 DNA in a cell-free system. The strengths of both these effects are determined by the number of direct repeats (DRs) present, and are also affected to similar degrees by different mutations within the repeated sequence. Several of the mutations were tested for their effect in cis on plasmid maintenance in the cell, and one was found to cause an increase in plasmid copy number. The results suggest that the direct repeats exert incompatibility by inhibiting DNA replication, presumably because they are the binding sites for a limiting essential protein.

Base Composition↗

PubMed1986

Characterization of the DNA of the hamster papovavirus. III. Mapping of inverted repeated DNA sequences within the viral genome.

Sequences with 2-fold axis of symmetry (inverted repeated sequences) have been detected and mapped on the hamster papovavirus (HaPV) genome by their ability to form secondary structures like hairpins or stem-loops on single-stranded HaPV DNA. DNA regions with secondary structure were visualized by electron microscopy and mapped according to the physical map of HaPV. Eleven positions containing inverted repeats could be determined. The nucleotide sequence within all of these inverted repeats may be related since these regions can crosshybridize with each other. The possible functions of these sequences within the HaPV genome are discussed with respect to conditions found for genomes like those from SV40 and polyoma virus to get first indications for the localization of DNA regions representing the origin and termination of replication of HaPV DNA.

Animals↗

PubMed1998

Extensive direct-tandem organization of a long repeat DNA sequence on the Y chromosome of chinook salmon (Oncorhynchus tshawytscha).

A long repetitive DNA sequence (OtY8) has been cloned from male chinook salmon and its genomic organization has been characterized. The repeat has a unit length of 8 kb and is present approximately 300 times per diploid male nucleus. All internal fragments within the 8-kb repeat segregate from father to son, suggesting that the entire repeat unit is located on the Y chromosome. The organization of this sequence into an 8-kb repeat unit is restricted to the Y chromosome, as are several male-specific repeat subtypes identified on the basis of restriction-site variation. The repeat possesses only weak internal sequence similarities, suggesting that OtY8 has not arisen by duplication of a smaller repeat unit, as is the case for other long tandem arrays found in eukaryotes. Based on a laddered pattern arising from partial digestion of genomic DNA with a restriction enzyme which cuts only once per repeat unit, this sequence is not dispersed on the Y chromosome but is organized as a head-to-tail tandem array. Pulse-gel electrophoresis reveals that the direct-tandem repeats are organized into at least six separate clusters containing approximately 12 to 250 copies, comprising some 2.4 Mb of Y-chromosomal DNA in total. Related sequences with nucleotide substitutions and DNA insertions relative to the Y-chromosomal fragment are found elsewhere in the genome but at much lower copy number and, although similar sequences are also found in other salmonid species, the amplification of the repeat into a Y-chromosome-linked tandem array is only observed in chinook salmon. The OtY8 repetitive sequence is genetically tightly associated with the sex-determination locus and provides an opportunity to examine the evolution of the Y chromosome and sex determination process in a lower vertebrate.

Animals↗

PubMed1986

Insertion and/or deletion of many repeated DNA sequences in human and higher ape evolution.

The total numbers of copies of two repeat families, L1 (Kpn I) and Alu, have been measured in the DNA of four higher apes by an accurate titration method. The number of members of the Alu family repeats in the four genomes are as follows: human, 910,000; chimpanzee, 330,000; gorilla, 410,000; orangutan, 580,000. For the Kpn I family (3'-ward higher frequency region) the number of copies in these genomes are as follows: human, 107,000; chimpanzee, 51,000; gorilla, 64,000; orangutan, 84,000. Thermal stability measurements show that, although the families of repeats are moderately divergent in sequence, little net sequence change has occurred during the evolution of the higher apes. Most or all of the members of these families of repeats are interspersed throughout the genome. Therefore, a large number of events of insertion and/or deletion of these DNA sequences has occurred during higher primate evolution.

Animals↗

PubMed1994

Mapping and organization of highly-repeated DNA sequences by means of simultaneous and sequential FISH and C-banding in 6x-triticale.

Three families of highly repeated sequences from rye and the rRNA multigenes (NOR and 5S) have been mapped by FISH and C-banding, in chromosomes of triticale. The pSc119.2 probe showed interstitial hybridization in chromosome arms 1RS, 1RL, 4RL, 5RL, 6RS, 6RL, 7RS and 7RL, and is very effective for chromosome identification of rye chromosomes in triticale. This sequence also hybridizes to the 4A, 5A and the seven B-genome wheat chromosomes. Simultaneous hybridization with the pSc119.2 and pTa794 (5S rRNA) is very useful to distinguish the metacentric chromosomes 2R and 3R. The pSc74 probe appears at interstitial sites in the long arm of the most heterobrachial chromosomes (5R and 6R). The three repetitive sequences of 120 bp, 480 bp, and 610 bp hybridize to telomeric regions in rye chromosomes. Different arrangements and complex organizations consisting of arrays of three or more family sequences were found. The results demonstrate a great variation in the relative arrangement of the repetitive sequences in the telomeres of the rye chromosomes. There were quantitative differences in each cytological marker between triticale lines in both in situ labelling and C-banding, probably as the result of differences in the number and/or kind of repeat sequence.

Chromosome Banding↗

PubMed1992

In vivo topoisomerase II cleavage of the Drosophila histone and satellite III repeats: DNA sequence and structural characteristics.

We have identified two classes of in vivo topoisomerase II cleavage sites in the Drosophila histone gene repeat. One class co-localizes with DNase I-hypersensitive regions and another novel class maps to a subset of consecutive nucleosome linker sites in the scaffold-associated region (SAR) of the histone gene loop. Prominent topoisomerase II cleavage is also observed in one of the linker regions of the two nucleosomes spanning satellite III, a centromeric SAR-like DNA sequence with a repeat length of 359 bp. At the sequence level, in vivo topoisomerase II cleavage is highly site specific. Comparison of 10 nucleosome linker sites defines an in vivo cleavage sequence whose major characteristic is a prominent GC-rich core. These GC-rich cleavage sites are flanked by extensive arrays of oligo(dA).oligo(dT) tracts characteristic of SAR sequences. Treatment of cells with distamycin selectively enhances cleavage at nucleosome linker sites of the SAR and satellite regions, suggesting that AT-rich sequences flanking cleavage sites may be involved in determining topoisomerase II activity in the cell. These observations provide evidence for the association of topoisomerase II with SARS in vivo.

Animals↗

PubMed1993

Deletion between directly repeated DNA sequences measured in extracts of bacteriophage T7-infected Escherichia coli.

An in vitro system based upon extracts of bacteriophage T7 infected Escherichia coli was used to study genetic deletions and to examine the importance of DNA replication in the deletion process. When T7 genomes with gene 1.3 inactivated by a 43-bp insert of random sequence DNA bracketed by 11-bp direct repeats were replicated in vitro the inserts were deleted with a frequency of about 10(-5) deletions per genome replication. Under conditions where deletion could take place only by recombination between direct repeats on distinct DNA molecules deletion frequency was at least an order of magnitude lower. These data demonstrate the utility of the in vitro system as a means for examining deletion mechanisms and underscore the importance of DNA replication in deletions.

Bacteriophage T7↗

PubMed2003

Tandemly repeated DNA sequences and centromeric chromosomal regions of Arabidopsis species.

Despite their common function, centromeric DNA sequences are not conserved between organisms. Most centromeres of animals and plants so far investigated have now been shown to consist of large blocks of tandemly repeated satellite sequences that are embedded in recombination-deficient heterochromatic regions. This central domain of satellite sequences that is postulated to mediate spindle attachment is surrounded by pericentromeric sequences incorporating various classes of repetitive sequences often including retroelements. The centromeric satellite DNA sequences are amongst the most rapidly evolving sequences and pose some fundamental problems of maintaining function. In this overview, we will discuss work on centromeric repetitive sequences in Arabidopsis thaliana and its relatives, and highlight some of the common features that are emerging when analysing closely related species.

Arabidopsis↗

PubMed2003

Molecular characterization of a family of tandemly repeated DNA sequences, TR-1, in heterochromatic knobs of maize and its relatives.

Two families of tandem repeats, 180-bp and TR-1, have been found in the knobs of maize. In this study, we isolated 59 clones belonging to the TR-1 family from maize and teosinte. Southern hybridization and sequence analysis revealed that members of this family are composed of three basic sequences, A (67 bp); B (184 bp) or its variants B' (184 bp), 2/3B (115 bp), 2/3B' (115 bp); and C (108 bp), which are arranged in various combinations to produce repeat units that are multiples of approximately 180 bp. The molecular structure of TR-1 elements suggests that: (1) the B component may evolve from the 180-bp knob repeat as a result of mutations during evolution; (2) B' may originate from B through lateral amplification accompanied by base-pair changes; (3) C plus A may be a single sequence that is added to B and B', probably via nonhomologous recombination; and (4) 69 bp at the 3' end of B or B', and the entire sequence of C can be removed from the elements by an unknown mechanism. Sequence comparisons showed partial homologies between TR-1 elements and two centromeric sequences (B repeats) of the supernumerary B chromosome. This result, together with the finding of other investigators that the B repeat is also fragmentarily homologous to the 180-bp repeat, suggests that the B repeat is derived from knob repeats in A chromosomes, which subsequently become structurally modified. Fluorescence in situ hybridization localized the B repeat to the B centromere and the 180-bp and TR-1 repeats to the proximal heterochromatin knob on the B chromosome.

Base Sequence↗

PubMed1985

Transcriptional measurements of mouse repeated DNA sequences.

We have carried out transcriptional measurements on several families of repeated sequences to define their expression in mouse cells. The majority of Alu family transcripts result from read-through from adjacent structural gene promoters while 20% are discrete RNA polymerase III products. Alu repeat members show preferential orientation within RNA polymerase II transcription units as evidenced by asymmetric representation of the complementary strands of the Alu family in hnRNA. We assessed whether 3 non-Alu repeated sequence families had their own promoters by strand symmetry measurements and size distribution analysis of repeat-homologous newly synthesized nuclear RNA. Transcription homologous to the R family is totally symmetric and is likely due to read-through from adjacent structural gene promoters. LLRep1 and Bam5 repeats, in contrast, exhibit consistent strand asymmetry which is suggestive that at least some members may be transcribed by their own promoters. Among 3 mouse tissues and 1 cultured cell line analyzed, no quantitative variation in the expression of any of these sequences was observed.

Animals↗

PubMed1991

A family of repeated DNA sequences in Toxoplasma gondii: cloning, sequence analysis, and use in strain characterization.

A Toxoplasma gondii genomic library was constructed in lambda EMBL3. Repeated fragments were detected by hybridization with radiolabeled total DNA from the parasite and one recombinant was chosen due to its strong hybridization signal. By using electrophoretic and hybridization analysis, four cross-hybridizating restriction fragments were selected and sequenced. The determined nucleotide sequence of these fragments (TGR1A, TGR1E, TGR2, and TGR4) has shown a complex system of conserved and degenerated repeats in which TGR1E corresponds to the most conserved element. This last sequence was used to investigate restriction fragment length polymorphisms among several T. gondii strains by Southern blotting.

Animals↗

PubMed1990

Preliminary characterization of the repeated DNA sequence from Vicia sativa.

We have identified a family of interspersed repeated sequences present in about 40,000 copies in the genomes of Vicia sativa and its near relative Vicia faba. The element vif is at least 6 kb in length, and members of the repeat family display a degree of heterogeneity in restriction pattern. Homologous elements in V. faba are much more heterogeneous than their V. sativa counterparts; however, analysis of five different V. faba lines revealed substantially the same patterns, suggesting that this family was reamplified prior to the divergence of the lines studied.

Cloning, Molecular↗