Molecular structure and chromosome distribution of three repetitive DNA families in Anemone hortensis L. (Ranunculaceae)

The structure, abundance and location of repetitive DNA sequences on chromosomes can characterize the nature of higher plant genomes. Here we report on three new repeat DNA families isolated from Anemone hortensis L.; (i) AhTR1, a family of satellite DNA (stDNA) composed of a 554–561 bp long EcoRV monomer; (ii) AhTR2, a stDNA family composed of a 743 bp long HindIII monomer and; (iii) AhDR, a repeat family composed of a 945 bp long HindIII fragment that exhibits some sequence similarity to Ty3/gypsy-like retroelements. Fluorescence in-situ hybridization (FISH) to metaphase chromosomes of A. hortensis (2n = 16) revealed that both AhTR1 and AhTR2 sequences co-localized with DAPI-positive AT-rich heterochromatic regions. AhTR1 sequences occur at intercalary DAPI bands while AhTR2 sequences occur at 8–10 terminally located heterochromatic blocks. In contrast AhDR sequences are dispersed over all chromosomes as expected of a Ty3/gypsy-like element. AhTR2 and AhTR1 repeat families include polyA- and polyT-tracks, AT/TA-motifs and a pentanucleotide sequence (CAAAA) that may have consequences for chromatin packing and sequence homogeneity. AhTR2 repeats also contain TTTAGGG motifs and degenerate variants. We suggest that they arose by interspersion of telomeric repeats with subtelomeric repeats, before hybrid unit(s) amplified through the heterochromatic domain. The three repetitive DNA families together occupy ∼10% of the A. hortensis genome. Comparative analyses of eight Anemone species revealed that the divergence of the A. hortensis genome was accompanied by considerable modification and/or amplification of repeats. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

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.     

Molecular organization of the Drosophila melanogaster Adh chromosomal region in D. replete and D. buzzatii, two distantly related species of the Drosophila subgenus

The molecular organization of a 1.944-Mb chromosomal region of Drosophila melanogaster around the Adh locus has been analyzed in two repleta group species: D. repleta and D. buzzatii. The extensive genetic and molecular information about this region in D. melanogaster makes it a prime choice for comparative studies of genomic organization among distantly related species. A set of 26 P1 phages from D. melanogaster were successfully hybridized using fluorescence in-situ hybridization (FISH) to the salivary gland chromosomes of both repleta group species. The results show that the Adh region is distributed in D. repleta and D. buzzatii over six distant sites of chromosome 3, homologous to chromosomal arm 2L of D. melanogaster (Muller's element B). This observation implies a density of 2.57 fixed breakpoints per Mb in the Adh region and suggests a considerable reorganization of this chromosomal element via the fixation of paracentric inversions. Nevertheless, breakpoint density in the Adh region is three times lower than that estimated for D. repleta chromosome 2, homologous to D. melanogaster 3R (Muller's element E). Differences in the rate of evolution among chromosomal elements are seemingly persistent in the Drosophila genus over long phylogenetic distances. This revised version was published online in July 2006 with corrections to the Cover Date.     

Molecular diversification of tandemly organized DNA sequences and heterochromatic chromosome regions in some triticeae species

The subtelomeric heterochromatin of rye (Secale cereale) chromosomes makes up 12–18% of the genome and consists largely of a small number of tandemly organized DNA sequence families. The genomic organization, chromosomal locations and the structural organization of monomer units of the major DNA sequences from these regions were investigated and compared in other Triticeae species from the generaSecale, Agropyron, Dasypyrum, Triticum andHordeum. Southern hybridization and polymerase chain reaction analysis established that all studied species preserve the tandem type of sequence organization but the copy number is altered drastically between species. In the pSc200 family, a fraction of the tandem arrays is present with a head-to-head orientation of dimers inS. cereale andS. montanum. Members of the same family are more heterogeneous and present as head-to-head monomers in theDasypyrum species andA. cristatum. In situ hybridization demonstrates different organization of the sequence families in the various species: pSc200 and pSc250 are concentrated in major blocks at the ends of most rye chromosome arms, whereas they are more dispersed and in smaller blocks inDasypyrum andAgropyron indicating that accumulation is not simply due to the sequence itself. In contrast to rye,D. villosum has large blocks of only pSc200 whereasD. breviaristatum shows greater amplification of pSc250. These data indicate that each repetitive family is an independent unit of evolution, and suggest that the twoDasypyrum species are not closely related. The data are discussed in terms of existing evolutionary models for repetitive DNA sequences. The contribution of random events, through molecular drive and selection, to the evolution of heterochromatic regions is considered.accepted for publication by S. Mizuno     

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.     

Molecular characterization of a centromeric satellite DNA in the hemiclonal hybrid frogRana esculenta and its parental species

Hybrid water frogsRana esculenta reproduce by hybridogenesis: one parental genome (ofRana lessonae) is excluded in the germ line, the other (ofRana ridibunda) is clonally transmitted to haploid gametes. The two parental species differ in that the amount of centromeric heterochromatin revealed by differential staining is much higher inRana ridibunda. An abundant, tandemly arrayed, centromeric satellite DNA, designated RrS1, is revealed inRana ridibunda genomes by the restriction endonucleaseStul, which generates a major repetitive sequence fragment of 300 and a minor one of 200 bp. This AT-rich (68%) satellite family is located at the centromeres of the five largest chromosomes (1–5) and of a medium to small heterobrachial one (8 or 9); it thus constitutes only part of the centromeric heterochromatin that characterizes allRana ridibunda chromosomes. RrS1 represents about 2.5% of the genome ofRana ridibunda; it may represent as little as 0.2% of the genome ofRana lessonae, and cannot be detected inXenopus laevis frogs orSalamandra salamandra andTriturus carnifex salamanders. Segments of the satellite sequence are similar to sequences of yeast centromeric DNA element CDEIII and of the mammalian CENP-B box. A role for RrS1 and other centromeric satellite DNAs in the germ line genome exclusion of the hybridogenetic frog hybrids, although suggested, has not yet been demonstrated.     

Divergence of satellite DNA and interspersion of dispersed repeats in the genome of the wild beet Beta procumbens

Several repetitive sequences of the genome of Beta procumbens Chr. Sm., a wild beet species of the section Procumbentes of the genus Beta have been isolated. According to their genomic organization, the repeats were assigned to satellite DNA and families of dispersed DNA sequences.The tandem repeats are 229–246 bp long and belong to an AluI restriction satellite designated pAp11. Monomers of this satellite DNA form subfamilies which can be distinguished by the divergence or methylation of an internal restriction site. The satellite is amplified in the section Procumbentes, but is also found in species of the section Beta including cultivated beet (Beta vulgaris). The existence of the pAp11 satellite in distantly related species suggests that the AluI sequence family is an ancient component of Beta genomes and the ancestor of the diverged satellite subfamily pEV4 in B. vulgaris. Comparative fluorescent in-situ hybridization revealed remarkable differences in the chromosomal position between B. procumbens and B. vulgaris, indicating that the pAp11 and pEV4 satellites were most likely involved in the expansion or rearrangement of the intercalary B. vulgaris heterochromatin.Furthermore, we describe the molecular structure, and genomic and chromosomal organization of two repetitive DNA families which were designated pAp4 and pAp22 and are 1354 and 582 bp long, respectively. The families consist of sequence elements which are widely dispersed along B. procumbens chromosomes with local clustering and exclusion from distal euchromatic regions. FISH on meiotic chromosomes showed that both dispersed repeats are colocalized in some chromosomal regions. The interspersion of repeats of the pAp4 and pAp22 family was studied by PCR and enabled the determination of repeat flanking sequences. Sequence analysis revealed that pAp22 is either derived from or part of a long terminal repeat (LTR) of an Athila-like retrotransposon. Southern analysis and FISH with pAp4 and pAp22 showed that both dispersed repeats are species-specific and can be used as DNA probes to discriminate parental genomes in interspecific hybrids. This was tested in the sugar beet hybrid PRO1 which contains a small B. procumbens chromosome fragment.     

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.     

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     

Chromosomal location and evolution of a satellite DNA family in seven sturgeon species

The Hind III satellite DNA family, isolated from the Acipenser naccarii genome, was used as a probe for fluorescent in-situ hybridization (FISH) on the karyotype of seven sturgeon species, six belonging to the genus Acipenser and one to Huso. All species except one (A. sturio) exhibit from 8 to 80 chromosome hybridization signals, mainly localized at the pericentromeric regions. Eight chromosomes with weak hybridization signals are present in H. huso and A. ruthenus, which are characterized by a karyotype with about 120 chromosomes. The species with 240–260 chromosomes, A. transmontanus, A naccarii, A. gueldenstaedtii, and A. baerii, show from 50 to 80 signals, prevalently localized around centromeres. Moreover, A. transmontanus and A. gueldenstaedtii show from 4 to 8 chromosomes with a double signal. The phylogenetic and evolutionary relationships among sturgeon species are discussed on the basis of number and morphology of signal-bearing chromosomes and on the localization of signals.     

Molecular and cytogenetic organization of the 5S ribosomal DNA array in chicken (Gallus gallus)

The 5S ribosomal (r) RNA genes encode a small (120-bp) highly-conserved component of the large ribosomal subunit. The objective of the present research was to study the molecular and cytogenetic organization of the chicken 5S rDNA. A predominant 2.2-kb gene (5S) consisting of a coding and intergenic spacer (IGS) region was identified in ten research and commercial populations. A variant gene repeat of 0.6kb (5S) was observed in some of the populations. Genetic linkage analysis and cytogenetic localization by fluorescence in-situ hybridization assigned the 5S rDNA to chromosome 9. The 5S rDNA array was determined to be 80.2±7.0kb upon electrophoretic sizing following EcoRV digestion. Sequence analysis of 5S IGS regions revealed considerable conservation between chicken subspecies (98.4% identity) as well as homology with vertebrate Pol III promoter and regulatory sequence motifs. Minor intraindividual sequence variation within 1000bp of IGS was observed in four cloned Red Jungle Fowl (Gallus gallus gallus) 5S repeats (95.5% identity in this region). Sequence comparisons between IGS regions of 5S and 5S genes indicated two short continuous (>20bp) and many short non-continuous homologous regions as well as other conserved features such as promoter and termination motifs.     

FISH to mitotic chromosomes and extended DNA fibres of Beta procumbens in a series of monosomic additions to beet (B. vulgaris)

The physical localization and organization of a Procumbentes-specific repetitive DNA sequence, PB6-4, on the chromosomes of Beta procumbens (2n=18) were studied, using FISH (fluorescence in situ hybridization) to mitotic chromosomes and extended DNA fibres. The chromosomes of B. procumbens were studied in metaphase complements of the species itself, as well as in preparations of a series of eight different B. procumbens-derived monosomic additions to B. vulgaris (2n=18). FISH to chromosome spreads of B. procumbens revealed that PB6-4 hybridizes to all chromosomes, predominantly in the pericentromeric regions, but with differences in size and brightness of the signals. Hybridization of PB6-4 to metaphase complements of B. vulgaris revealed no signals, indicating that cross-hybridization with the genome of this species was negligible. Consequently, hybridization of PB6-4 to metaphase complements of the monosomic additions yielded fluorescent signals on the alien chromosomes only. The previously observed differences in size and brightness of the fluorescent spots were confirmed using the single alien chromosomes. FISH of PB6-4 to extended DNA fibres of the monosomic additions indicated differences in the fluorescent track lengths between the alien chromosomes. Measurements of the fluorescent tracts allowed classification into discrete groups, varying from one to three groups per B. procumbens chromosome. The data revealed that the brightness or size of the signal at mitotic metaphase and the length of the fluorescent tracks on the DNA fibres were correlated. This revised version was published online in July 2006 with corrections to the Cover Date.     

The chloroplast trnP-trnW-petG gene cluster in the mitochondrial genomes of Beta vulgaris,B. trigyna and B. webbiana: evolutionary aspects

The chloroplast trnP-trnW-petG gene cluster has been identified in the mitochondrial DNA (mtDNA) of sugar beet (Beta vulgaris). The chloroplast-derived trnW gene is transcribed in the mitochondria; the other two genes, however, do not seem to be transcribed. This gene cluster is also present in the mitochondrial genomes of two wild Beta species, B. trigyna and B. webbiana. Sugar beet and the two wild relatives share 100% sequence identity in the coding regions of both the mitochondrial trnP and trnW genes. On the other hand, the petG genes from the wild Beta mtDNAs were found to be disrupted either by a 5-bp duplication (B. trigyna) or by a deletion of the 5 region (B. webbiana). A data-base search revealed that a conserved sequence of 60 bp is present in the trnP-trnW intergenic region of the mitochondrial genomes of the three Beta species as well as in other higher plants, including wheat and maize, and that the conserved sequence is absent from the chloroplast counterpart. Our results thus favour the hypothesis of a monophyletic origin of the trnP-trnW-petG cluster found in the plant mitochondrial genomes examined.     

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     

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.     

Direct detection of repetitive,whole chromosome paint and telomere DNA probes by immunogold electron microscopy

Biotinylated repetitive, whole chromosome paint and telomere DNA probes were investigated at the electron microscope level after non-isotopicin situ hybridization and direct immunogold detection. The protocol described allowed the visualization of a biotinylated chromosome 1 specific satellite DNA probe in the light microscope without silver intensification. This sensitive method was exploited to analyse factors contributing to signal strength in immunogold chromosome painting. Furthermore, it allowed us to investigate the distribution of (TTAGGG)n telomere repeats in human metaphase chromosomes and interphase nuclei. Telomeric and internal (TTAGGG)n repeats were detected at high spatial resolution in human metaphase chromosomes. In the periphery of lymphocyte interphase nuclei, two types of telomere hybridization signals were observed. They differed remarkably in compactness, indicating two types of chromatin conformation present at the interphase telomeresin situ.     

Sequence analysis, chromosomal distribution and long-range organization show that rapid turnover of new and old pBuM satellite DNA repeats leads to different patterns of variation in seven species of the Drosophila buzzatii cluster

We aimed to study patterns of variation and factors influencing the evolutionary dynamics of a satellite DNA, pBuM, in all seven Drosophila species from the buzzatii cluster (repleta group). We analyzed 117 alpha pBuM-1 (monomer length 190 bp) and 119 composite alpha/beta (370 bp) pBuM-2 repeats and determined the chromosome location and long-range organization on DNA fibers of major sequence variants. Such combined methodologies in the study of satDNAs have been used in very few organisms. In most species, concerted evolution is linked to high copy number of pBuM repeats. Species presenting low-abundance and scattered distributed pBuM repeats did not undergo concerted evolution and maintained part of the ancestral inter-repeat variability. The alpha and alpha/beta repeats colocalized in heterochromatic regions and were distributed on multiple chromosomes, with notable differences between species. High-resolution FISH revealed array sizes of a few kilobases to over 0.7 Mb and mutual arrangements of alpha and alpha/beta repeats along the same DNA fibers, but with considerable changes in the amount of each variant across species. From sequence, chromosomal and phylogenetic data, we could infer that homogenization and amplification events involved both new and ancestral pBuM variants. Altogether, the data on the structure and organization of the pBuM satDNA give insights into genome evolution including mechanisms that contribute to concerted evolution and diversification. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.