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PubMed2005

Novel repeated DNA sequences in safflower (Carthamus tinctorius L.) (Asteraceae): cloning, sequencing, and physical mapping by fluorescence in situ hybridization.

Two novel repetitive DNA sequences, pCtKpnI-1 and pCtKpnI-2, were isolated from Carthamus tinctorius (2n = 2x = 24) and cloned. Both represent tandemly repeated sequences. The pCtKpnI-1 and pCtKpnI-2 clones constitute repeat units of 343-345 bp and 367 bp, respectively, with 63% sequence heterogeneity between the two. Fluorescence in situ hybridization (FISH) was employed on metaphase chromosomes of C. tinctorius using, simultaneously, pCtKpnI-1 and pCtKpnI-2 repeated sequences. The pCtKpnI-1 sequence was found to be exclusively localized at subtelomeric regions on most of the chromosomes. On the other hand, sequence of the pCtKpnI-2 clone was distributed on two nucleolar and one nonnucleolar chromosome pairs. The satellite, and the intervening chromosome segment between the primary and secondary constrictions, in the two nucleolar chromosome pairs were wholly constituted by pCtKpnI-2 repeated sequence. The pCtKpnI-2 repeated sequence, showing partial homology to intergenic spacer (IGS) of 18S-25S ribosomal RNA genes of an Asteraceae taxon (Centaurea stoebe), and the 18S-25S rRNA gene clusters were located at independent, but juxtaposed sites in the nucleolar chromosomes. Variability in the number, size, and location of the two repeated sequences provided identification of most of the chromosomes in the otherwise not too distinctive homologues within the complement. This article reports the start of a molecular cytogenetics program targeting the genome of safflower, a major world oil crop about whose genetics very little is known.

Base Sequence↗
PubMed1982

Rearrangement of repeated DNA sequences during development of macronucleus in Tetrahymena thermophila.

Three clones of non-repetitive sequences and six clones containing repetitive sequences were obtained from micronuclear DNA of Tetrahymena thermophila. All the non-repetitive and three repetitive sequences had the same organization in micro- and macronuclear DNAs as revealed by blot hybridization. On the other hand, the remaining three clones with repetitive sequences had apparently different organization in the two nuclear DNAs. All these repetitive sequences showed a smear on the blot in addition to a number of discrete bands when micronuclear DNA was digested with EcoR I. In macronuclear DNAs, these sequences invariably became one or two bands and the smear disappeared. We conclude that, when a macronucleus develops from a micronucleus, the non-repetitive sequences amplify by more than 20 times with relatively few rearrangement, whereas some selected portions of repeated and/or repeat-contiguous sequences are amplified with rather extensive reorganization.

Animals↗
PubMed2006

Bacterial contingency loci: the role of simple sequence DNA repeats in bacterial adaptation.

Bacterial pathogens face stringent challenges to their survival because of the many unpredictable, often precipitate, and dynamic changes that occur in the host environment or in the process of transmission from one host to another. Bacterial adaptation to their hosts involves either a mechanism for sensing and responding to external changes or the selection of variants that arise through mutation. Here we review how bacterial pathogens exploit localized hypermutation, through polymerase slippage of simple sequence repeats (SSRs), to generate phenotypic variation and enhanced fitness. These SSRs are located within the reading frame or in the promoter of a subset of genes, often termed contingency loci, whose functions are usually involved in direct interactions with host structures.

Adaptation, Physiological↗
PubMed1997

Distribution of highly repeated DNA sequences in species of the genus Secale.

The presence and distribution of the most important highly repetitive DNA sequences of rye in cultivated and wild species of the genus Secale were investigated using fluorescence in situ hybridization. Accurate identification of individual chromosomes in the most commonly recognized species or subspecies of the genus Secale (S. cereale, S. ancestrale, S. segetale, S. afghanicum, S. dighoricum, S. montanum, S. montanum ssp. kuprijanovii, S. africanum, S. anatolicum, S. vavilovii, and S. silvestre) was achieved using three highly repetitive rye DNA sequences (probes pSc119.2, pSc74, and pSc34) and the 5S ribosomal DNA sequence pTa794. It is difficult to superimpose trends in the complexity of repetitive DNA during the evolution of the genus on conclusions from other cytogenetic and morphological assays. However, there are two clear groups. The first comprises the self-pollinated annuals S. silvestre and S. vavilovii that have few repeated nucleotide sequences of the main families of 120 and 480 bp. The second group presents amplification and interstitialization of the repeated nucleotide sequences and includes the perennials S. montanum, S. anatolicum, S. africanum, and S. kuprijanovii, as well as the annual and open-pollinated species S. cereale and its related weedy forms. The appearance of a new locus for 5S rRNA in S. cereale and S. ancestrale suggests that cultivated ryes evolved from this wild weedy species.

Biological Evolution↗
PubMed2003

Copro-diagnosis of Echinococcus granulosus infection in dogs by amplification of a newly identified repeated DNA sequence.

Diagnosis of Echinococcus granulosus infection in dogs by detecting adult worms recovered post mortem or purged from the intestines after treatment with arecoline is not suitable for mass screening. Large-scale diagnosis by detection of copro-antigens is useful but only with relatively high intensity infections, and only by genus. To provide a more sensitive and specific diagnosis, a polymerase chain reaction (PCR) assay was developed, that amplified a target repeated sequence (EgG1 Hae III) newly identified in the genome of the common sheep strain of E. granulosus. This repeated sequence consists of approximately 6,900 copies, arranged in tandem, in groups of 2-6 repeats. The corresponding primers used in the PCR easily detected a single egg with no cross-amplification of DNA from closely related cestodes, including E. multilocularis and Taenia spp. Fecal samples from naturally infected dogs, with 2-10,000 E. granulosus worms at necropsy, were all PCR positive, while E. multilocularis or Taenia spp. positive controls as well as non-endemic controls were all PCR negative. This copro-PCR assay was demonstrated to be 100% specific and also detected all necropsy-positive E. granulosus-infected dogs. It is suggested that this copro-PCR assay has the potential for pre-mortem diagnosis of E. granulosus infection even in areas where E. granulosus and E. multilocularis are co-endemic.

Animals↗
PubMed1983

Kpn I family of long interspersed repeated DNA sequences in primates: polymorphism of family members and evidence for transcription.

An approximately equal to 2-kilobase-pair-long member (Kpn I-LS1) of the African green monkey Kpn I family of repeated sequences has been cloned, subjected to sequence analysis, and compared to other family members which are over 6 kilobase pairs (Kpn I-alpha 7) and 829 base pairs (Kpn I-RET) long. Both Kpn I-LS1 and Kpn I-RET lack sequences found at the ends of the longer family member and their structures resemble those of processed genes. Kpm I-LS1 sequences are colinear with part of the long family member, Kpn I-alpha 7. However, although all sequences in Kpn I-RET are represented in Kpn I-LS1, the two are not colinear; Kpn I-RET is missing 731 base pairs found in Kpn I-LS1 and one segment flanking the deletion is inverted. The results demonstrate that Kpn I family members are not only of different lengths but may also contain scrambled arrangements of common sequences. Sequences in Kpn I-LS1 hybridize to RNA from monkey and human cells, indicating that some family members are transcribed.

Animals↗
PubMed2001

Instability of bacterial artificial chromosome (BAC) clones containing tandemly repeated DNA sequences.

The cloning and propagation of large DNA fragments as bacterial artificial chromosomes (BACs) has become a valuable technique in genome research. BAC clones are highly stable in the host, Escherichia coli, a major advantage over yeast artificial chromosomes (YACs) in which recombination-induced instability is a major drawback. Here we report that BAC clones containing tandemly repeated DNA elements are not stable and can undergo drastic deletions during routine library maintenance and DNA preparation. Instability was observed in three BAC clones from sorghum, rice, and potato, each containing distinct tandem repeats. As many as 46% and 74% of the single colonies derived from a rice BAC clone containing 5S ribosomal RNA genes had insert deletions after 24 and 120 h of growth, respectively. We also demonstrated that BAC insert rearrangement can occur in the early stage of library construction and duplication. Thus, a minimum growth approach may not avoid the instability problem of such clones. The impact of BAC instability on genome research is discussed.

Chromosomes, Artificial, Bacterial↗
PubMed1996

Complex arrangement of dispersed repeated DNA sequences in Oryza officinalis.

A 525-bp BglII fragment was isolated from Oryza officinalis DNA (accession W1278) and shown to correspond to a new dispersed repetitive DNA sequence with specificity restricted to a subset of the wild rice with a C genome. The sequence of the fragment was determined but it does not correspond to any sequence already present in databases. It contains several imperfect palindromes. Larger genomic clones (12-18 kbp) were isolated and all contain sequences homologous to the BglII element. Analysis of these clones confirms that the BglII element is dispersed in the O. officinalis genome. From one genomic clone, the sequences adjacent to the BglII element were subcloned and used as probes to demonstrate that the sequences flanking the BglII element are variable in different genomic clones and that some of them are also dispersed repetitive sequences. The genomic specificity of two of these dispersed repeats was evaluated and shown to be different from that of the initial BglII element. This analysis revealed a complex arrangement of various dispersed repeated sequences.

Bacterial Proteins↗
PubMed2004

The genomic organization and evolutionary distribution of a tandemly repeated DNA sequence family in the genus Crocus (Iridaceae).

From a recombinant DNA-library from Crocus vernus, two closely related clones of highly repetitive DNA, pCvKB7 and pCvKB8, were sequenced and their genomic distribution and organization were investigated by Southern and in situ hybridization. The lengths of the clones were 181 and 178 bp respectively; the sequences were approximately 85% identical, and thus belonged to a sequence family, named the pCvKB8-family. No homologous sequences were found in the databases (BLAST made may 2004). The presence of pCvKB8 in 52 Crocus species and six species from other genera were analyzed by Southern hybridization. The sequence family was essentially Crocus-specific. However, the distribution of hybridization signal across the genus showed poor agreement with the taxonomic structure of the Crocus genus, suggesting that the subdivision does not follow the phylogeny of this sequence family. The chromosomal distribution on three Crocus species was essentially identical: tandem organization close to all telomeres and most centromeres, with a few additional intercalary sites.

Crocus↗
PubMed1996

A variable region of Anticarsia gemmatalis nuclear polyhedrosis virus contains tandemly repeated DNA sequences.

A variable region (PstI-T fragment) of two genotypic variants of the Anticarsia gemmatalis multiple nucleocapsid nuclear polyhedrosis virus (AgMNPV) was sequenced and compared. This region, which is known to have deletions and duplications in AgMNPV variants, was shown to be formed of units of a 127 bp tandemly repeated sequence containing two 30 bp imperfect palindromes. Southern blot experiments showed that the PstI-T fragment contained one of the four homologous regions mapped interspersed in the AgMNPV genome. The comparison of the nucleotide sequences of the two variants, AgMNPV-2D and AgMNPV-D7, showed that the difference between these two variants in one of these regions (hr4) was caused by the addition or elimination of 381 bp corresponding exactly to three of the 127 bp repeated sequences. An analysis of the sequence showed homology to the homologous regions (hr) of other baculoviruses. The sequence upstream of the repetitive region contained a sequence homologous to the N-terminal portion of the Autographa californica MNPV and Choristoneura fumiferana MNPV p74 gene.

Animals↗
PubMed1993

Typing of artiodactyl MHC-DRB genes with the help of intronic simple repeated DNA sequences.

An efficient oligonucleotide typing method for the highly polymorphic MHC-DRB genes is described for artiodactyls like cattle, sheep and goat. By means of the polymerase chain reaction, the second exon of MHC-DRB is amplified as well as part of the adjacent intron containing a mixed simple repeat sequence. Using this primer combination we were able to amplify the MHC-DRB exons 2 and adjacent introns from all of the investigated 10 species of the family of Bovidae and giraffes. Therefore, the DRB genes of novel artiodactyl species can also be readily studied. Oligonucleotide probes specific for the polymorphisms of ungulate DRB genes are used with which sequences differing in at least one single base can be distinguished. Exonic polymorphism was found to be correlated with the allele lengths and the patterns of the repeat structures. Hence oligonucleotide probes specific for different simple repeats and polymorphic positions serve also for typing across species barriers. The strict correlation of sequence length and exonic polymorphism permits a preselection of specific oligonucleotides for hybridization. Thus more than 20 alleles can already be differentiated from each of the three species.

Alleles↗
PubMed1995

Repeated DNA sequences in mycobacteria.

In tuberculosis, it is often important to establish the source of infection and to determine whether disease is due to a new strain of Mycobacterium tuberculosis or to relapse. To cope with the resurgence of tuberculosis and atypical mycobacterioses in AIDS patients, on the one hand, and to overcome the limitations of classical bacteriological procedures on the other, the development of rapid, sensitive, and reliable diagnostic and epidemiologic tools is highly desirable. Molecular typing methods are often based on repeated genes such as those for rRNA. Ribotyping is of limited use with pathogenic mycobacteria, as the slow-growers possess a single rRNA operon, while the fast-growers have two. This problem has been overcome by the discovery and study of repeated DNA elements in mycobacterial genomes, as these provide an alternative pathway for diagnostic and epidemiological investigations.

Base Sequence↗
PubMed1986

Linkage studies of polymorphic, repeated DNA sequences in centromeric regions of human chromosomes.

The DNA at human centromeric regions was characterized by using a repetitive sequence, 308, which localizes in situ exclusively to centromeres of all chromosomes. We previously noted that this sequence is enriched on chromosome 6 and has chromosome-specific organization on 6, 3, 7, 14, X, and Y. In addition to this basic organization, sequences homologous to 308 are polymorphic among normal individuals. The variants are transmitted in a Mendelian manner within a family. To determine the chromosome origin of the variants, we studied their linkage to markers of various chromosomes. Linkage analysis of one pedigree segregating two polymorphisms shows that the 2.6-kilobase (kb) BamHI and 2.6-kb TaqI fragments are linked to each other and to the HLA loci on chromosome 6. Data from another family shows that 2.8-kb TaqI, 4.0-kb TaqI, and 1.3-kb BamHI polymorphic fragments are linked and are probably near the Fy locus on chromosome 1. By dot blot analysis, we determined that the relative amount of these sequences in the genome is not measurably different between unrelated individuals. Thus, the polymorphisms represent changes in homologous 308 sequences on specific chromosomes and can be used as chromosome-specific markers. Linkage studies using polymorphisms of repeated sequences will be most useful within a kindred, especially from an inbred population, because polymorphic repeats of the same restriction size may be heterogeneous in origin.

Centromere↗
PubMed2002

Characterization of a novel hexameric repeat DNA sequence in the promoter of the immediate early gene, IEX-1, that mediates 1alpha,25-dihydroxyvitamin D(3)-associated IEX-1 gene repression.

1alpha,25-Dihydroxyvitamin D(3)(1alpha,25(OH)(2)D(3)), the active metabolite of vitamin D(3), mediates anti-proliferative effects in cells by regulating the expression of 1alpha,25(OH)(2)D(3)-responsive genes. The expression of the proliferation-promoting Immediate Early gene X-1 (IEX-1) is reduced by 1alpha,25(OH)(2)D(3) through unknown mechanisms. Here we report the presence of a novel inhibitory hexameric repeat DNA response element in the promoter of the human IEX-1 gene that mediates 1alpha,25(OH)(2)D(3)-associated IEX-1 gene repression. To localize a vitamin D sensitive DNA response element we transfected the keratinocyte-like cell line, HaCaT, (referred as HaCaT) with a series of plasmids containing full-length and truncated IEX-1 promoter elements fused to the luciferase reporter gene in the absence or presence of 1alpha,25(OH)(2)D(3), and we performed electrophoretic gel mobility assays in the presence of receptors for 1alpha,25(OH)(2)D(3) (vitamin D receptor, VDR) and 9-cis-retinoic acid (RXRalpha). We mapped a negative response element between nt -405 and -391(15 bp) of theIEX-1 promoter (5'-TGAACC AGG GAGTCA-3') that mediates transcriptional inhibition in response to 1alpha,25(OH)(2)D(3) and which requires expression of both nuclear receptors for 1alpha,25(OH)(2)D(3) and 9-cis-retinoic acid. Our data indicate that the physiological repression of IEX-1 gene expression by 1alpha,25(OH)(2)D(3) is directly mediated by nuclear VDR/RXRalpha heterodimers through a specific transcriptional element.

Apoptosis Regulatory Proteins↗
PubMed1985

Organization of Plasmodium falciparum genome: 1. Evidence for a highly repeated DNA sequence.

Plasmodium falciparum DNA, isolated from the merozoite stage, was cleaved with HindIII and cloned in pBR322 and lambda L47.1 vectors. Plasmid clones containing 13.4, 7.0, 4.3, 4.1 and 1.5 kb inserts were characterized in some detail. The inserts contain several repeating units of smaller size. Nucleic acid hybridization studies showed that the repeat element is present in the Plasmodium DNA at a very high copy number and appears to be distributed widely throughout the genome.

Animals↗
PubMed1995

Repeated DNA sequences isolated by microdissection. I. Karyotyping of barley (Hordeum vulgare L.).

We report on microdissection, cloning and sequence, and Southern and fluorescence in situ hybridization (FISH) analysis of one moderately and one highly amplified repetitive DNA element, pHvMWG2314 and pHvMWG2315, respectively, isolated from barley (Hordeum vulgare L.) chromosome arm 3HL. The pHvMWG2315 sequence hybridizes to all 14 telomeric or subtelomeric regions of the barley chromosomes as determined by FISH. The 50 different hybridization sites that include intercalary signals allow the discrimination of all 14 chromosome arms and the construction of a kariotype of barley. The tandemly repeated subtelomeric element of 331 bp exists in all Triticeae species tested (H. vulgare, Agropyron elongatum, Secale cereale, Triticum tauschii, T. turgidum, and T. aestivum). It is AT rich (66%), exibits 84% sequence homology to subfragments of the D genome ¿specific¿ 1-kb element pAs1 of T. tauscii and 75% homology to interspersed genome-specific DNA sequence pHcKB6 from H. chilence. The repetitive sequence pHvMWG2314 is moderately amplified in barley and highly amplified in hexaploid wheat. The in situ experiments revealed no distinct signals on barley chromosomes, indicating a dispersed character for the sequence. The significance of the results for the identification of chromosomes and chromosome aberrations in FISH experiments are discussed.

Base Composition↗
PubMed1983

A second major class of Alu family repeated DNA sequences in a primate genome.

We have analyzed repetitive DNA sequences in a prosimian, Galago crassicaudatus, and found that there are two distinct, highly repetitive families of sequences related to the human Alu family. The Type I family is closely analogous to the human Alu Family. The Type II family of repeats, which appears to be present in higher copy number, has a right half that is almost identical to the Type I family. However, the left half of the Type II sequence shows only limited homology to the galago Type I or human Alu families. A comparison of homologous sequences in the left half indicated that they are centered in regions of the Alu family which function as RNA polymerase III promoters. We have also observed at least one example of a Type II left half that was integrated into the genome independent of the Alu family right half sequence. The Type II family appears to be of much more recent evolutionary origin than the Type I and may have arisen by the independent integration of a RNA polymerase III promoter adjacent to the right half of a Type I Alu family sequence.

Animals↗