Molecular characterization and physical localization of highly repetitive DNA sequences from Brazilian Alstroemeria species

Highly repetitive DNA sequences were isolated from genomic DNA libraries of Alstroemeria psittacina and A. inodora. Among the repetitive sequences that were isolated, tandem repeats as well as dispersed repeats could be discerned. The tandem repeats belonged to a family of interlinked Sau3A subfragments with sizes varying from 68–127 bp, and constituted a larger HinfI repeat of approximately 400 bp. Southern hybridization showed a similar molecular organization of the tandem repeats in each of the Brazilian Alstroemeria species tested. None of the repeats hybridized with DNA from Chilean Alstroemeria species, which indicates that they are specific for the Brazilian species. In-situ localization studies revealed the tandem repeats to be localized in clusters on the chromosomes of A. inodora and A. psittacina: distal hybridization sites were found on chromosome arms 2PS, 6PL, 7PS, 7PL and 8PL, interstitial sites on chromosome arms 2PL, 3PL, 4PL and 5PL. The applicability of the tandem repeats for cytogenetic analysis of interspecific hybrids and their role in heterochromatin organization are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

A novel family of repetitive DNA sequences amplified site-specifically on the W chromosomes in Neognathous birds

A novel family of repetitive DNA sequences was molecularly cloned from ApaI-digested genomic DNA of two Galliformes species, Japanese quail (Coturnix japonica) and guinea fowl (Numida meleagris), and characterized by chromosome in-situ hybridization and filter hybridization. Both the repeated sequence elements produced intensely painted signals on the W chromosomes, whereas they weakly hybridized to whole chromosomal regions as interspersed-type repetitive sequences. The repeated elements of the two species had high similarity of nucleotide sequences, and cross-hybridized to chromosomes of two other Galliformes species, chicken (Gallus gallus) and blue-breasted quail (Coturnix chinensis). The nucleotide sequences were conserved in three other orders of Neognathous birds, the Strigiformes, Gruiformes and Falconiformes, but not in Palaeognathous birds, the Struthioniformes and Tinamiformes, indicating that the repeated sequence elements were amplified on the W chromosomes in the lineage of Neognathous birds after the common ancestor diverged into the Palaeognathae and Neognathae. They are components of the W heterochromatin in Neognathous birds, and a good molecular cytogenetic marker for estimating the phylogenetic relationships and for clarifying the origin of the sex chromosome heterochromatin and the process of sex chromosome differentiation in birds.     

Isolation, characterization and chromosome localization of repetitive DNA sequences in bananas (Musa spp.)

Partial genomic DNA libraries were constructed in Musa acuminata and M. balbisiana and screened for clones carrying repeated sequences, and sequences carrying rDNA. Isolated clones were characterized in terms of copy number, genomic distribution in M. acuminata and M. balbisiana, and sequence similarity to known DNA sequences. Ribosomal RNA genes have been the most abundant sequences recovered. FISH with probes for DNA clones Radka1 and Radka7, which carry different fragments of Musa 26S rDNA, and Radka14, for which no homology with known DNA sequences has been found, resulted in clear signals at secondary constrictions. Only one clone carrying 5S rDNA, named Radka2, has been recovered. All remaining DNA clones exhibited more or less pronounced clustering at centromeric regions. The study revealed small differences in genomic distribution of repetitive DNA sequences between M. acuminata and M. balbisiana, the only exception being the 5S rDNA where the two Musa clones under study differed in the number of sites. All repetitive sequences were more abundant in M. acuminata whose genome is about 12% larger than that of M. balbisiana. While, for some sequences, the differences in copy number between the species were relatively small, for some of them, e.g. Radka5, the difference was almost thirty-fold. These observations suggest that repetitive DNA sequences contribute to the difference in genome size between both species, albeit to different extents. Isolation and characterization of new repetitive DNA sequences improves the knowledge of long-range organization of chromosomes in Musa. This revised version was published online in July 2006 with corrections to the Cover Date.     

Organization and chromosomal location of repetitive DNA sequences in three species of squamate reptiles

Repetitive DNA sequences were isolated from the genomes of species representing three major clades of squamate reptiles. A repetitive sequence (Cn4C7) was isolated from the New Mexican whiptail lizard,Cnemidophorus neomexicanus. This sequence is distributed throughout the chromosomes, but is more concentrated in the telomeric region. Cn4C7 also hybridizes to the chromosomes of otherCnemidophorus. Some evidence was found for concerted evolution of this repeat in hybrid unisexual lineages. In the lesser earless lizard,Holbrookia maculata, the predominant repeat in the genome is represented by a sequence (Hm1E11) which is restricted to the area flanking the centromere in all species ofHolbrookia. Two families of repetitive sequences (one dispersed, and the other telomeric) were isolated from the western diamondback rattlesnake,Crotalus atrox. The type and distribution of repetitive sequences in squamates is often taxon-specific, and may be useful as characters for elucidating taxonomic relationships.     

Palindromic unit highly repetitive DNA sequences exhibit species specificity within Enterobacteriacea

Palindromic units (PU, or REP for repetitive extragenic palindrome) constitute a family of DNA sequences of 40 nucleotides which is highly repeated in the genome of Escherichia coli. We analysed the presence of PU sequences in 99 different bacterial genomes by cross-hybridization. When PU sequences were used as a probe, only DNA from Enterobacteriaceae closely related to E. coli exhibited an appreciable hybridization signal: Shigella sonnei, Shigella boydii, Salmonella enteritica serotype Typhimurium, Citrobacter freundii and Levinea malonatica. Furthermore, these bacteria could be divided into two groups which corresponded to a slight difference in their PU sequence: the E. coli group includes S. sonnei and S. boydii; the S. enteritica serotype Typhimurium group includes C. freundii and L. malonatica.     

The position of repetitive DNA sequence in the southern cattle tick genome permits chromosome identification

Fluorescent in-situ hybridization (FISH) using meiotic chromosome preparations and highly repetitive DNA from the southern cattle tick, Rhipicephalus microplus, was undertaken to investigate genome organization. Several classes of highly repetitive DNA elements were identified by screening a R. microplus bacterial artificial chromosome (BAC) library. A repeat unit of approximately 149 bp, RMR-1 was localized to the subtelomeric regions of R. microplus autosomes 1–6 and 8–10. A second repeat unit, RMR-2 was localized to the subtelomeric regions of all autosomes and the X chromosome. RMR-2 was composed of three distinct repeat populations, RMR-2a, RMR-2b and RMR-2c of 178, 177 and 216 bp in length, respectively. Localization of an rDNA probe identified a single nucleolar organizing region on one autosome. Using a combination of labeled probes, we developed a preliminary karyotype for R. microplus. We present evidence that R. microplus has holocentric chromosomes and explore the implications of these findings for tick chromosome biology and genomic research. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effect of a new antitumor drug cycloplatam on DNA structure and production

The new antitumor drug cycloplatam [amine (cyclopentylamine)-S-(-)-malatoplatinum(II)] caused dose-dependent formation of interstrand DNA ligationsin vitro depending on the duration of incubation. Like cisplatin, cycloplatam caused a deep and long inhibition of DNA production in tumor cells. The effect of cycloplatam in bone marrow and small intestine epithelium cells was weaker and shorter than that of cisplatin. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 125, No. 2, pp. 197–199, February, 1998     

FISH mapping and molecular organization of the major repetitive sequences of tomato

This paper presents a bird’s-eye view of the major repeats and chromatin types of tomato. Using fluorescence in-situ hybridization (FISH) with Cot-1, Cot-10 and Cot-100 DNA as probes we mapped repetitive sequences of different complexity on pachytene complements. Cot-100 was found to cover all heterochromatin regions, and could be used to identify repeat-rich clones in BAC filter hybridization. Next we established the chromosomal locations of the tandem and dispersed repeats with respect to euchromatin, nucleolar organizer regions (NORs), heterochromatin, and centromeres. The tomato genomic repeats TGRII and TGRIII appeared to be major components of the pericentromeres, whereas the newly discovered TGRIV repeat was found mainly in the structural centromeres. The highly methylated NOR of chromosome 2 is rich in [GACA]₄, a microsatellite that also forms part of the pericentromeres, together with [GA]₈, [GATA]₄ and Ty1-copia. Based on the morphology of pachytene chromosomes and the distribution of repeats studied so far, we now propose six different chromatin classes for tomato: (1) euchromatin, (2) chromomeres, (3) distal heterochromatin and interstitial heterochromatic knobs, (4) pericentromere heterochromatin, (5) functional centromere heterochromatin and (6) nucleolar organizer region.     

Comparative genomic in situ hybridization (cGISH) analysis on plant chromosomes revealed by labelled Arabidopsis DNA

A new approach for comparative cytogenetic banding analysis of plant chromosomes has been established. The comparative GISH (cGISH) technique is universally applicable to various complex genomes of Monocotyledonae (Triticumaestivum, Agropyronelongatum, Secalecereale, Hordeumvulgare, Alliumcepa, Muscariarmenaticum and Liliumlongiflorum) and Dicotyledonae (Viciafaba, Betavulgaris, Arabidopsisthaliana). Labelled total genomic DNA of A. thaliana generates signals at conserved chromosome regions. The nucleolus organizing regions (NORs) containing the majority of tandemly repeated rDNA sequences, N-band regions containing satellite DNA, conserved homologous sequences at telomeres and additional chromosome-characteristic markers were detected in heterologous FISH experiments. Multicolour FISH analysis with repetitive DNA probes simultaneously revealed the chromosome assignment of 56 cGISH signals in rye and 61 cGISH signals in barley. Further advantages of this technique are: (1) the fast and straightforward preparation of the probe; (2) the generation of signals with high intensity and reproducibility even without signal amplification; and (3) no requirement of species-specific sequences suitable for molecular karyotype analysis. Hybridization can be performed without competitive DNA. Signal detection without significant background is possible under low stringency conditions. The universal application of this fast and simple one-step fluorescence banding technique for plant cytogenetic and plant genome evolution is discussed.     

Chromosomal location of endogenous geminivirus-related DNA sequences inNicotiana tabacum L.

TheN. tabacum (tobacco) nuclear genome carries approximately 25 multiple direct repeats of a geminivirus-related DNA (GRD) sequence that probably arose by illegitimate recombination, following geminivrus infection, duringNicotiana evolution. Each GRD repeat carries sequences similar to the geminiviralAL1 gene of the tomato golden mosaic virus (TGMV), encoding a protein required for viral DNA replication, plus thecis-essential replication origin. Using a cloned 14-kb GRD repeat sequence as a probe for fluorescencein situ hybridization (FISH), we identified a unique tobacco chromosome carrying GRD. Translocations between chromosomes of the tobacco S and T genomes were used as physical markers by sequentially hybridizing chromosomes with labelled GRD and total genomic DNA fromN. sylvestris (equivalent to the S genome). The 25S, 18S and 5.8S ribosomal gene clusters were detected in double-labelling experiments for use as additional markers to identify the chromosomal location of GRD. GRD occupies one site on a homologous pair of small submetacentrics from the T genome characterized by a lack of either translocated segments from the S genome or ribosomal genes. GRD provides an additional marker for the small chromosomes of the T genome and a useful phylogenetic tool.     

Cloning and genetic analyses of two highly polymorphic,moderately repetitive nuclear DNAs from Phytophthora infestans

Summary Randomly selected clones from a Phytophthora infestans partial genomic library were characterized by hybridizing individual clones to Southern blots of total genomic DNA digested with the restriction enzyme EcoRI. Among 59 clones that were screened on seven different central-Mexican isolates, five revealed a unique banding pattern for each isolate tested. Two of these clones were tested further; the banding patterns produced by both were somatically stable when probed to DNA from 63 single-zoospore (asexual) progeny from five different parent isolates. For one probe, RG57, each band appeared to represent a unique genetic locus in three different crosses, and each locus segregated for the presence or absence of a band. No bands were found to be allelic, but two pairs of cosegregating loci were identified. Genetic analyses of the other probe (RG7) revealed many more pairs of cosegregating bands and some bands which were allelic. When these probes were hybridized to DNA from the other five species in Phytophthora group IV, probe RG57 hybridized strongly to DNA from P. colocasiae, P. phaseoli and P. mirabilis, but weakly or not at all to that of P. hibernalis and P. ilicis. Probe RG7 hybridized fairly strongly to DNA from all six species. Because the sequence recognized by probe RG57 appears to be evolutionarily conserved, and is dispersed, moderately repetitive and highly polymorphic, it could be very useful in additional studies on the genetics and population biology of P. infestans.     

Characterization of four novel genomic regions of uropathogenic Escherichia coli highly associated with the extraintestinal virulent phenotype: a jigsaw puzzle of genetic modules

Extraintestinal pathogenic Escherichia coli (ExPEC) are a major cause of urinary tract infections, sepsis, and neonatal meningitis. A variety of virulence factors in these strains is encoded by mobile genetic elements, such as transposons or pathogenicity islands (PAIs). Using subtractive cloning of ExPEC genomes, we recently detected short DNA fragments, which were significantly associated with the extraintestinal virulent phenotype. In this study, we identified four novel genomic DNA regions of the highly virulent uropathogenic E. coli strain JS299 carrying these previously identified DNA fragments. Characterization of the partial sequences of the genomic DNA regions revealed complex DNA arrangements with variable genetic compositions regarding the G+C contents and codon usage patterns. The prevalence of 15 previously uncharacterized genes was determined in a collection of clinical ExPECs and commensal E. coli strains by means of DNA microarray analyses. From this, 13 novel DNA sequences were demonstrated to be significantly associated with extraintestinal virulent strains, and thus may represent new virulence traits. Beside genes predicted to play a role in metabolic functions, such as sucrose utilization (scr), we identified DNA sequences shared by both ExPEC and enteropathogenic E. coli (EPEC). These sequences were significantly more prevalent among ExPECs when compared to commensal E. coli isolates. Our results support the idea of a considerable genetic variability among ExPEC strains and suggest that the novel genomic determinants described in this study may contribute to the ExPEC virulence.     

The conserved fraction of repetitive sequences in the human and mammalian genome in health,in pathology,and for long-term mutagenic exposure

The percentage and the size distribution of weakly divergent sites of repetitive sequences of human chromosomal DNA were determined in health and in hereditary genome diseases which are characterized by chromosome instability and predisposition to malignant tumor. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 117, N ^o 1, pp. 85–88, January, 1994 Presented by A. I. Archakov, Member of the Russian Academy of Medical Sciences     

Evidence for a sequence-directed conformation periodicity in the genomic highly repetitive DNA detectable with single-strand-specific chemical probe potassium permanganate

A single-strand-specific chemical probe, potassium permanganate (KMnO₄), was used to study the sequence-dependent conformation periodicity of tandem multicopy repetitive DNA sequences HRS60 and GRS (Nicotiana species) at the level of single base pair and dinucleotide step. Local DNA structures, sensitive to KMnO₄, revealed periodicity of 182±2 bp, equal to the length of repeat units. Permanganate-sensitive local structures were mapped in both DNA strands of genomic HRS60 sequences and were found to be linked to d(A)_n tracts. These adenine tracts are located in the proximity of the intrinsically curved domains. Distamycin A increased reactivity of the DNA but decreased the specificity of DNA cleavage. Similar conformation periodicity has been detected also in the canrep family of repeats (Brassica species). All studied repetitive sequences are predominantly located in the constitutive heterochromatin. We discuss the role of conformation periodicties in relation to a structural code for nucleosome phasing at tandem arrays of DNA repeats.accepted for publication by J.S. (Pat) Heslop-Harrison     

Molecular and cytogenetic characterization of repetitive DNA in the Antarctic polyplacophoran Nuttallochiton mirandus

Two highly repeated DNAs, designated NmE1/NmE2 and NmE5, were identified by EcoRV digestion in the chiton Nuttallochiton mirandus (Mollusca: Polyplacophora). The comparison of the sequences obtained showed high similarity in 5' and 3' regions and the NmE5 sequence displayed an inserted sequence that might arise from a transposable element. Southern blotting analyses suggested a tandem organization of both satellite DNA families identified. Moreover, dot blot analyses, performed on several molluscan species, revealed a different degree of conservation of the repeated DNAs. Fluorescence in-situ hybridizations (FISH) on metaphase chromosomes showed that both satellite DNAs are located at centromeric regions.     

Significance of specific protein S-S bonds in the structural-functional organization of DNA

The role of S-S bridges of residual protein in the structural organization of DNA is investigated. The effects of various S-S splitting agents on the naturally occurring DNA-RP protein complexes isolated from various eukaryotic and prokaryotic cells are studied. It is demonstrated that, depending on the incubation conditions, thiols induce dissociation of the DNA-RP complexes to double-strand fragment-DNA subunits of varied size. It is found that the DNA-RP complexes contain specific S-S bonds that may determine different levels of DNA organization in the cromosome. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 118, N ^o 8, pp. 180–183, August, 1994 Presented by N. N. Trapeznikov, Member of the Russian Academy of Medical Sciences     

Structural organization of the genome of the zygomycete Absidia glauca: evidence for high repetitive DNA content

Summary Total cell DNA of Absidia glauca has a GC-content of 44.6% ± 0.5% as determined from optical melting profiles which is in good accordance with values from equilibrium centrifugation in bisbenzimide containing CsCl gradients (46.2% = 1.1%), whereas mitochondrial DNA has a GC-content of only 30%. The genome size of Absidia glauca is approximately 36,000 kb, 8.6 times that of Escherichia coli. Three kinetically different fractions could be identified in reassociation experiments: a foldback-DNA fraction, comprising approximately 10% of the total DNA, repetitive DNA (25%) and single copy DNA (65%). This relatively high amount of repetitive DNA could partly be ascribed to ribosomal DNA (13%) and a new interspersed repetitive element (rAg1) which has been cloned in pBR325.     

Physical mapping of repetitive DNA sequences and 5S and 18S–26S rDNA in five wild species of the genusHordeum

The genetic relationships between several wild species and subspecies of the genusHordeum were assessed using fluorescencein situ hybridization (FISH). Plant material included natural populations of wild barley growing in Spain of the annual species,H. marinum ssp.marinum (2n=14) andgussoneanum (2n=14), andH. murinum ssp.murinum (2n=28), andleporinum (2n=28) and the perennial speciesH. bulbosum (2n=14) andH. secalinum (2n=28), plus the South American perennial speciesH. chilense (2n=14). FISH was used to locate the chromosomal sites of two rDNA multigene families 5S and 18S–26S (pTa71 and pTa794) and three repetitive DNA sequences (pSc119.2, pAs1 and pHch950) isolated from different species and genera. The seven chromosomes of the diploid species were readily distinguished by their external morphology and hybridization patterns to pTa71, pTa794, pSc119.2 and pAs1. These DNA probes were also useful for the identification of homologous chromosomes and in differentiating these from unidentified chromosomes in the tetraploid taxa. The use of the probe pHch950 permitted intergenomic differentiation in tetraploids and supports the diphyletic origin ofH. murinum andH. secalinum. Thein situ experiments yielded the following conclusions: (1) differences between the subspeciesmarinum andgussoneanum; (2) close relationships between the subspeciesmurinum andLeporinum; and (3) major differences in physical mapping betweenH. bulbosum and the remaining taxa. The genomic and phylogenetic relationships between taxa, as inferred from the results, are discussed.accepted by J.S. (Pat) Heslop-Harrison     

Whole-comparative genomic hybridization (W-CGH): 1. The quick overview of repetitive DNA sequences on a genome

We present a technique (W-CGH) based on Comparative Genomic Hybridization (CGH), but using whole DNA probes, which permits the identification of chromosomal polymorphisms related to highly repetitive DNA sequences that exist between the two genomes compared. The procedure employs two differently colored whole DNA probes from two different individuals that are mixed and hybridized to metaphase chromosomes. The method provides a simple way to map whole genome differences for highly repetitive DNA sequences between two individuals, since it does not require chromosome-specific probes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Integrative transformation of a zygomycete,Absidia glauca,with vectors containing repetitive DNA

Summary Experimental evidence for integration of transformed DNA into the genome of Absidia glauca, a member of the fungal class of zygomycetes is presented. According to the limited knowledge on the molecular biology of these fungi, autonomous replication of transformed plasmids seems to be the preferential mode of DNA propagation. By inserting fragments of highly repetitive DNA elements into an autonomously replicating vector conferring neomycin resistance, we were able to obtain integrative transformation events. With such plasmids we observed stable mitotic propagation of a selective marker gene (NPT II under the control of a homologous actin promoter). Analysis of DNA from transformants in Southern type experiments, as well as restriction analysis of retransformants into Escherichia coli, provide evidence that integration of foreign DNA into the genome of Absidia glauca is possible. These transformation events are often associated with the appearance of mutant phenotypes.