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PubMed2006

Characterization of Erwinia amylovora strains from different host plants using repetitive-sequences PCR analysis, and restriction fragment length polymorphism and short-sequence DNA repeats of plasmid pEA29.

AIMS: The three main aims of the study were the assessment of the genetic relationship between a deviating Erwinia amylovora strain isolated from Amelanchier sp. (Maloideae) grown in Canada and other strains from Maloideae and Rosoideae, the investigation of the variability of the PstI fragment of the pEA29 plasmid using restriction fragment length polymorphism (RFLP) analysis and the determination of the number of short-sequence DNA repeats (SSR) by DNA sequence analysis in representative strains. METHODS AND RESULTS: Ninety-three strains obtained from 12 plant genera and different geographical locations were examined by repetitive-sequences PCR using Enterobacterial Repetitive Intergenic Consensus, BOX and Repetitive Extragenic Palindromic primer sets. Upon the unweighted pair group method with arithmetic mean analysis, a deviating strain from Amelanchier sp. was analysed using amplified ribosomal DNA restriction analysis (ARDRA) analysis and the sequencing of the 16S rDNA gene. This strain showed 99% similarity to other E. amylovora strains in the 16S gene and the same banding pattern with ARDRA. The RFLP analysis of pEA29 plasmid using MspI and Sau3A restriction enzymes showed a higher variability than that previously observed and no clear-cut grouping of the strains was possible. The number of SSR units reiterated two to 12 times. The strains obtained from pear orchards showing for the first time symptoms of fire blight had a low number of SSR units. CONCLUSIONS: The strains from Maloideae exhibit a wider genetic variability than previously thought. The RFLP analysis of a fragment of the pEA29 plasmid would not seem a reliable method for typing E. amylovora strains. A low number of SSR units was observed with first epidemics of fire blight. SIGNIFICANCE AND IMPACT OF THE STUDY: The current detection techniques are mainly based on the genetic similarities observed within the strains from the cultivated tree-fruit crops. For a more reliable detection of the fire blight pathogen also in wild and ornamentals Rosaceous plants the genetic features of deviating E. amylovora strains have to be studied in detail.

Culture Media↗
PubMed1991

The effect of the length of direct repeats and the presence of palindromes on deletion between directly repeated DNA sequences in bacteriophage T7.

The frequency of genetic deletion between directly repeated DNA sequences in bacteriophage T7 was measured as a function of the length of the direct repeat. The non-essential ligase gene (gene 1.3) of bacteriophage T7 was interrupted with pieces of synthetic DNA bracketed by direct repeats of various lengths. Deletion of these 76 bp long inserts was too low to be measured when the direct repeats were less than 6 bp long. However, the frequency of deletion of inserts with longer direct repeats increased exponentially as the length of the repeats increased from 8 to 20 bp. When inverted repeats (palindromes) were designed in the midst of the insert there was essentially no increase in deletion frequency between 10 bp direct repeats. But, the same palindromic sequences increased the deletion frequency between 5 bp direct repeats by at least two orders of magnitude. Thus, in this system homology at the endpoints is a more important determinant of deletion frequency than is the presence of palindromes between the direct repeats.

Base Sequence↗
PubMed1983

Tandemly repeated DNA sequences from Xenopus laevis. I. Studies on sequence organization and variation in satellite 1 DNA (741 base-pair repeat).

A family of tandemly repeated sequences, having a basic repeating unit of 741 base-pairs, has been identified in Xenopus laevis DNA and designated satellite 1. Apart from its rather long repeat unit, the characteristics of this DNA appear to be quite similar to those of complex satellite DNAs from other organisms. The nucleotide sequence of a cloned repeat unit shows no evidence of simpler internal repeats, and there is no obvious suggestion of reasonable RNA- or protein-coding regions. Transcripts homologous to this DNA could not be demonstrated in liver, embryo or oocyte RNAs. By blot-hybridization, satellite 1 has been shown to exist in the genome chiefly as tandem repeats of the 741 base-pair sequence. However, there are a number of repeats that differ from the normal sequence (as judged by loss or gain of restriction sites) and some that differ in length. Similar variants are often, but not always, clustered. Characterization of genomic clones of this satellite has confirmed the tandem organization and clustering of variants. The nature of some variants has been elucidated in more detail. Some regions of the basic repeat seem to be more prone to variation than are others.

Animals↗
PubMed1986

The evolution of restricted recombination and the accumulation of repeated DNA sequences.

We suggest hypotheses to account for two major features of chromosomal organization in higher eukaryotes. The first of these is the general restriction of crossing over in the neighborhood of centromeres and telomeres. We propose that this is a consequence of selection for reduced rates of unequal exchange between repeated DNA sequences for which the copy number is subject to stabilizing selection: microtubule binding sites, in the case of centromeres, and the short repeated sequences needed for terminal replication of a linear DNA molecule, in the case of telomeres. An association between proximal crossing over and nondisjunction would also favor the restriction of crossing over near the centromere. The second feature is the association between highly repeated DNA sequences of no obvious functional significance and regions of restricted crossing over. We show that highly repeated sequences are likely to persist longest (over evolutionary time) when crossing over is infrequent. This is because unequal exchange among repeated sequences generates single copy sequences, and a population that becomes fixed for a single copy sequence by drift remains in this state indefinitely (in the absence of gene amplification processes). Increased rates of exchange thus speed up the process of stochastic loss of repeated sequences.

Animals↗
PubMed1993

Isolation of Trypanosoma cruzi specific nuclear repeated DNA sequences.

We described the isolation of two genomic fragments, called E12 and E22, containing highly repeated DNA sequences from Trypanosoma cruzi. The E12 and E22 fragments have molecular sizes of about 1.1 and 0.8 Kb, respectively. Copy number determinations indicate that each one of these sequences are repeated about 5 x 10(3) times in the parasite genome. E12 and E22 elements do not cross-hybridize indicating that they are not related in nucleotide sequence. The Southern blot analysis of total DNA digested with a variety of restriction enzymes shows the existence of a complex pattern of hybridization bands with the repeated elements indicating that they are interspersed along the T. cruzi genome. In fact, pulsed field gel electrophoresis together with hybridization evidenced that both sequences are present in all or nearly all the chromosomal bands. Clones bearing these repetitive elements could be useful for diagnostic of the Chagas' disease and strain classification purposes.

Animals↗
PubMed1996

First isolation of tandemly repeated DNA sequences in New World vultures and phylogenetic implications.

A highly repeated DNA sequence composed of closely related subunits that ranged from 171 to 176 base pairs has been cloned and characterized in the king vulture (Sarcoramphus papa). Related sequences were also isolated in the black vulture (Coragyps atratus). This new family of avian repetitive DNA elements is here termed the "HaeIII family." Genomic DNAs from a number of avian species were probed with one of the king vulture restriction fragments. In the cathartids, the hybridization patterns showed no individual or sexual variations. A strong HaeIII ladder was present in the two aforementioned species as well as in the Andean condor (Vultur gryphus), but in the black vulture the bands of the ladder alternated in intensity. Weaker hybridization signals were obtained in two ciconids, the jabiru stork (Jabiru mycteria) and the white stork (Ciconia ciconia). The HaeIII repeat was not detected in accipitrid birds of prey, a Polyborinae falconid, pelecanids, and psittacids.

Animals↗
PubMed2000

The chromosome complement of Olea europaea L.: characterization by differential staining of the chromatin and in-situ hybridization of highly repeated DNA sequences.

The chromosome complement of olive (Olea europaea L.) has been characterized by differential staining of the chromatin and chromosomal localization of highly repeated DNA sequences and ribosomal cistrons. DAPI staining produces different-sized positive bands in various locations on all the chromosomes. By combining this band pattern with the results obtained from cytological hybridization of OeTaq80, OeTaq178, and OeGEM86 DNA tandem repeats, most of the pairs can be distinguished from each other, in spite of the large number of chromosomes (2n = 46), their small size and similar morphology. Different tandem-repeated DNA sequences may be contained into single heterochromatic chromosome regions, even though there are regions where repeats of only one family are present. OeTaq80- and OeGEM86-related DNA sequences are rather specific to the heterochromatin at the chromosome ends, while most sequences related to the longer OeTaq178 probe are confined to interstitial heterochromatin. Some exceptions suggest that major chromosomal rearrangements occurred during genome evolution. Polymorphism, which may differentiate olive cultivars, was observed within chromosome pairs I, V, and VII.

Chromatin↗
PubMed1996

An approach for identifying simple sequence repeat DNA polymorphisms near cloned cDNAs and genes. Linkage studies of the islet amyloid polypeptide/amylin and liver glycogen synthase genes and NIDDM.

Genetic factors contribute to the development of NIDDM, and genes involved in regulating pancreatic beta-cell function and insulin's effects on glucose metabolism are good candidates for being NIDDM susceptibility loci. However, testing candidate genes for linkage to NIDDM depends on the identification of highly informative DNA polymorphisms in or near the candidate locus. Here we describe an approach for identifying highly polymorphic markers near candidate genes that utilizes the emerging physical map of the human genome. A sequence-tagged site from the candidate gene is used to screen the Centre d'Etude du Polymorphisme Humain megabase-insert yeast artificial chromosome library, which contains information on the physical localization of >3,000 genetically mapped simple sequence repeat DNA polymorphisms. Thus, identification of a yeast artificial chromosome containing the candidate locus will in many instances also identify a physically linked simple sequence repeat DNA polymorphism that can be used as a marker for the candidate gene in linkage studies. We have used this approach to identify a marker for the islet amyloid polypeptide gene on chromosome 12. The physical mapping of this gene to a yeast artificial chromosome showed that it was in the same yeast artificial chromosome as the gene encoding liver glycogen synthase, another possible NIDDM susceptibility gene. Affected sib pair studies using a simple sequence repeat DNA polymorphism physically linked to the islet amyloid polypeptide and liver glycogen synthase genes showed no evidence for linkage with NIDDM, indicating that they are not major genes contributing to NIDDM susceptibility.

Amyloid↗
PubMed1989

Identification of a DNA fragment in the genome of Bordetella pertussis carrying repeated DNA sequences also present in other Bordetella species.

A DNA fragment (3.8 kbp) which hybridized to repeated sequences in the genome of Bordetella species has been cloned from Bordetella pertussis chromosomal DNA. Eleven subclones were constructed from this fragment. They exhibited distinct inter-species hybridization patterns in genomic blots of each of the Bordetella used in the study. One subclone revealed intraspecies variability among B. pertussis strains and another did not hybridize to B. parapertussis. The 3.8 kbp DNA fragment possesses a middle sequence surrounded by repeated sequences organized in an opposite symmetrical orientation. The external inverted repeats of it hybridized to a 680 bp DNA sequence which is located about 800 bp upstream of the pertussis toxin operon. The novel structural organization of the 3.8 kbp fragment suggests the possibility that this DNA segment is an IS-like element which may have an important function in the expression of virulence determinants in Bordetella bacteria.

Blotting, Southern↗
PubMed1988

Structure of a hypervariable tandemly repeated DNA sequence on the short arm of the human Y chromosome.

The structure of a repeated DNA sequence located on the short arm of the human Y chromosome is described. Genomic mapping and cloning in lambda or cosmid vectors show that the repeated sequence consists of units 20.3 x 10(3) base-pairs long that contain the three previously described DNA sequences: Y-156, Y-190 and Y-223a. Analysis of male genomic DNA by pulsed-field gel electrophoresis shows that the units are tandemly arranged and are organized into two blocks. The major block is hypervariable in size and alleles in the range approximately 540 x 10(3) to 800 x 10(3) base-pairs were detected. The minor block is not variable in size and is approximately 60 x 10(3) base-pairs long. Analysis of rearranged Y chromosomes shows that both blocks are located on the short arm of the chromosome. Most commonly, the major block is distal to the minor block, but the opposite arrangement is also found.

Cell Line↗
PubMed1990

A novel avian W chromosome DNA repeat sequence in the lesser black-backed gull (Larus fuscus).

The phenol emulsion reassociation technique was used to isolate and clone a female specific, repetitive DNA sequence from Larus fuscus. The repeat, designated P2000-17, is restricted to the W chromosome, although related sequences occur elsewhere in the genome of L. fuscus. Similar sequences were detected in the genome of six other bird species from outside the genus Laridae, but the sequence occurs less frequently and to a similar extent in both sexes. The 298 bp DNA sequence of P2000-17 was determined and found to have extensive sequence identity to the rabbit dihydropyridine (DHP) receptor calcium channel. P2000-17 is represented once within a larger 8.6 kb tandem repeat (LfW-1), which has a complex internal DNA sequence. LfW-1 is highly conserved between repeat motifs and may comprise 3% of the female genome. The possible evolutionary origin of LfW-1 is discussed in relation to the repeat types found on the W and Y chromosomes of other species.

Animals↗
PubMed1997

Organisation and molecular analysis of repeated DNA sequences in the rice blast fungus Magnaporthe grisea.

The distribution of a previously described repeated DNA sequence present as a 1.3-kb PstI fragment in the genome of the rice blast fungus Magnaporthe grisea was analysed by carrying out DNA fingerprint analysis of 36 isolates including rice, non-rice and laboratory strains. The analysis of various higher-molecular-weight PstI fragments with homology to the 1.3-kb repeat revealed that these may arise predominantly from transposon insertions or point mutations. Analysis of a 5.1-kb derivative revealed both a point mutation at a PstI site and an insertion of a putative transposable element which caused an increase in molecular weight from 1.3 to 5.1 kb. Another repeat element of 1.4 kb was identified and found to exist in association with the 1.3-kb repeat. Both 1.3- and 1.4-kb elements were found to be parts of MGR583 (Hamer et al. 1989), a LINE-like element. These elements were present in a high copy number in all the rice and a majority of non-rice pathogens indicating that MGR583 is not a host-specific sequence as reported earlier. Our results suggest that repeated DNA elements in M. grisea have amplified independently of one another and further indicate that different isolates of M. grisea may have evolved from several distinct lines of origin.

Ascomycota↗
PubMed1993

Digital codes from hypervariable tandemly repeated DNA sequences in the Plasmodium falciparum circumsporozoite gene can genetically barcode isolates.

DNA typing systems currently used in parasitology involve either hybridising Southern blots with repetitive sequence probes or amplifying genomic sequences using the polymerase chain reaction (PCR). Both such approaches assay allelic length variation, usually in unexpressed tandemly repeated DNA sequences. Where an appropriate target locus exists, an alternative PCR-based strategy which reveals allelic sequence variation in tandemly repeated DNA offers a more accurate and internally controlled assay. We describe such a strategy for the rapid extraction of information on tandem repeat sequence variation from hypervariable alleles, and apply it to the Plasmodium falciparum CS gene. The extreme variability of such DNA 'barcodes' can be used to identify parasite stocks and lineages. This system is also potentially useful for population genetic and epidemiological studies since it offers the possibility of following the spread of distinctively marked parasite genotypes in samples taken from infected individuals.

Animals↗
PubMed2004

A repeat DNA sequence from the Y chromosome in species of the genus Microtus.

In most mammals, the Y chromosome is composed of a large amount of constitutive heterochromatin. In some Microtus species, this feature is also extended to the X chromosome, resulting in enlarged (giant) sex chromosomes. Several repeated DNA sequences have been described in the gonosomal heterochromatin of these species, indicating that it has heterogeneous and species-specific composition and distribution. We have cloned an AT-rich, 851-bp long, repeated DNA sequence specific for M. cabrerae Y chromosome heterochromatin. The analysis of other species of the genus Microtus indicated that this sequence is also located on the Y chromosome (male-specific) in three species (M. agrestis, M. oeconomus and M. nivalis), present on both Y and X chromosomes and on some autosomes in M. arvalis and absent in the genome of M. guentheri. Our data also suggest that the mechanism of heterochromatin amplification operating on the sex chromosomes could have been different in each species since the repeated sequences of the gonosomal heterochromatic blocks in M. cabrerae and M. agrestis are different. The absence of this sequence in the mouse genome indicates that its evolutionary origin could be recent. Future analysis of the species distribution, localization and sequence of this repeat DNA family in arvicolid rodent species could help to establish the unsolved phylogenetic relationships in this rodent group.

Animals↗
PubMed2001

Characterization of a highly repeated DNA sequence family in five species of the genus Eulemur.

The karyotypes of Eulemur species exhibit a high degree of variation, as a consequence of the Robertsonian fusion and/or centromere fission. Centromeric and pericentromeric heterochromatin of eulemurs is constituted by highly repeated DNA sequences (including some telomeric TTAGGG repeats) which have so far been investigated and used for the study of the systematic relationships of the different species of the genus Eulemur. In our study, we have cloned a set of repetitive pericentromeric sequences of five Eulemur species: E. fulvus fulvus (EFU), E. mongoz (EMO), E. macaco (EMA), E. rubriventer (ERU), and E. coronatus (ECO). We have characterized these clones by sequence comparison and by comparative fluorescence in situ hybridization analysis in EMA and EFU. Our results showed a high degree of sequence similarity among Eulemur species, indicating a strong conservation, within the five species, of these pericentromeric highly repeated DNA sequences.

Animals↗
PubMed1997

Variable (CA/GT)n simple sequence repeat DNA in the alga Chlamydomonas.

A partial genomic DNA library of Chlamydomonas reinhardtii was screened with an (AC)11 probe for the presence of (CA/GT)n simple sequence repeats (SSRs). Based on the frequency of these repeats in the partial genomic library, we estimate that (CA/GT)n repeats occur at a rate of about one every 17.7 kb in the C. reinhardtii genome. Ten positive clones were sequenced and four polymerase chain reaction (PCR) primer sets flanking (CA/GT)n sequences were constructed for four loci. The PCR was used to specifically amplify these regions from multiple isolates of C. reinhardtii. All four loci were highly polymorphic in the C. reinhardtii isolates. A simple Mendelian inheritance pattern was found for all four loci, which showed 2:2 segregation in the tetrads resulting from a cross between C. reinhardtii and C. smithii. Our results suggest that these simple sequence repeat DNA loci will be useful for identity testing, population studies, linkage analysis, and genome mapping in Chlamydomonas.

Animals↗
PubMed1989

Rapid methods to visualize Y-specific repeated DNA sequences in cytological preparations.

We described rapid methods to detect Y-specific repeated DNA sequences in cytological preparations using in situ hybridization. A human Y chromosome specific DNA probe with an insert equivalent to that in pHY2.1 was labelled with [alpha-32P]dCTP or photobiotin, and hybridized to chromosome preparations. Signals were visualized specifically on Y chromosomes after 1 day's autoradiography or a couple of hours treatment with streptavidin alkaline phosphatase/BCIP/NBT. These methods are useful for molecular confirmation of Y-autosomal translocations.

DNA↗
PubMedDate not supplied

Characterization of MboI repeat DNA sequence of Anopheles stephensi.

MboI repeat fragment of mosquito Anopheles stephensi has been isolated by molecular cloning. The restriction map and entire nucleotide sequence of the 433bp insert has been determined. Hybridization of this repeat DNA with restriction enzyme digest of mosquito DNA does not show an interspersed pattern but suggests that this repeat may be tandemly repeated at one major site and a few minor sites in the genome of Anopheles stephensi. The hybridization pattern also indicates that this repeat family comprises of many similar but non-identical sequences. An open reading frame encoding 66 amino acids with an initiation and two tandem termination codons has been identified. This putative 66 amino acid polypeptide sequence has significant homology to a small region of RNA tumour viral envelope protein.

Animals↗