Chromosome-specific organization of human alpha satellite DNA

Restriction endonuclease analysis of human genomic DNA has previously revealed several prominent repeated DNA families defined by regularly spaced enzyme recognition sites. One of these families, termed alpha satellite DNA, was originally identified as tandemly repeated 340- or 680-base pair (bp) EcoRI fragments that hybridize to the centromeric regions of human chromosomes. We have investigated the molecular organization of alpha satellite DNA on individual human chromosomes by filter hybridization and in situ hybridization analysis of human DNA and DNA from rodent/human somatic cell hybrids, each containing only a single human chromosome. We used as probes a cloned 340-bp EcoRI alpha satellite fragment and a cloned alpha satellite-containing 2.0-kilobase pair (kbp) BamHI fragment from the pericentromeric region of the human X chromosome. In each somatic cell hybrid DNA, the two probes hybridized to a distinct subset of DNA fragments detected in total human genomic DNA. Thus, alpha satellite DNA on each of the human chromosomes examined--the X and Y chromosomes and autosomes 3, 4, and 21--is organized in a specific and limited number of molecular domains. The data indicate that subsets of alpha satellite DNA on individual chromosomes differ from one another, both with respect to restriction enzyme periodicities and with respect to their degree of sequence relatedness. The results suggest that some, and perhaps many, human chromosomes are characterized by a specific organization of alpha satellite DNA at their centromeres and that, under appropriate experimental conditions, cloned representatives of alpha satellite subfamilies may serve as a new class of chromosome-specific DNA markers.     

Pericentric satellite DNA sequences in Pipistrellus pipistrellus (Vespertilionidae; Chiroptera)

This paper reports the molecular and cytogenetic characterization of a HindIII family of satellite DNA in the bat species Pipistrellus pipistrellus. This satellite is organized in tandem repeats of 418 bp monomer units, and represents approximately 3% of the whole genome. The consensus sequence from five cloned monomer units has an A-T content of 62.20%. We have found differences in the ladder pattern of bands between two populations of the same species. These differences are probably because of the absence of the target sites for the HindIII enzyme in most monomer units of one population, but not in the other. Fluorescent in situ hybridization (FISH) localized the satellite DNA in the pericentromeric regions of all autosomes and the X chromosome, but it was absent from the Y chromosome. Digestion of genomic DNAs with HpaII and its isoschizomer MspI demonstrated that these repetitive DNA sequences are not methylated. Other bat species were tested for the presence of this repetitive DNA. It was absent in five Vespertilionidae and one Rhinolophidae species, indicating that it could be a species/genus specific, repetitive DNA family.     

Molecular characterization and chromosomal localization of two families of satellite DNA in Prochilodus lineatus (Pisces,Prochilodontidae), a species with B chromosomes

Prochilodus lineatus, an abundant species in the Mogi-Guaçu river basin, represents a large part of the region's fishing potential. Karyotypic analyses based on classic cytogenetic techniques have revealed the presence of 54 meta-submetacentric type chromosomes, together with the occurrence of small supernumerary chromosomes with intra and interindividual variations. This paper describes the genomic organization of two families of satellite DNA in the P. lineatus genome. The chromosomal localization these two repetitive DNA families through fluorescence in situ hybridization (FISH) demonstrated that the SATH1 satellite DNA family, composed of approximately 900 bp, was located in the pericentromeric region of a group of chromosomes of the standard complement, as well as on all the B chromosomes. The SATH2 satellite family has a monomeric unit of 441 bp and was located in the pericentromeric regions of some chromosomes of the standard complement, but was absent in the B chromosomes. Double FISH analyses showed that these two families participate jointly in the pericentromeric organization of several chromosomes of this species. The data obtained in this study support the hypothesis that the B chromosomes derive from chromosomes of the standard complement, which are carriers of the SATH1 satellite DNA.     

Characterization and chromosome location of satellite DNA in the leaf beetle Chrysolina americana (Coleoptera, Chrysomelidae)

This paper is the first record of the satellite DNA of the specialized phytophagous genus Chrysolina. The satellite DNA of Chrysolina americana is organized in a tandem repeat of monomers 189 bp long, has a A + T content of 59.6 % and presents direct and inverted internal repeats. Restriction analysis of the total DNA with methylation sensitive enzymes suggests that this repetitive DNA is undermethylated. In situ hybridization with a biotinylated probe of the satellite DNA showed the pericentromeric localization of these sequences in all meiotic bivalents. The presence of this repetitive DNA in other species of the genus was also tested by Southern analysis. The results showed that this satellite DNA sequence is specific to the C. americana genome and has not been found in three other species of Chrysolina with a different choice of host plants than in the former. This revised version was published online in July 2006 with corrections to the Cover Date.     

Sequence subfamilies of satellite repeats related to rDNA intergenic spacer are differentially amplified on<Emphasis Type="Italic"> Vicia sativa</Emphasis> chromosomes

We cloned and sequenced the Vicia sativa 25S-18S rDNA intergenic spacer (IGS) and the satellite repeat S12, thought to be related to the spacer sequence. The spacer was shown to contain three types of subrepeats (A, B, and C) with monomers of 173 bp (A), 10 bp (B), and 66 bp (C), separated by unique or partially duplicated sequences. Two spacer variants were detected in V. sativa that differed in length (2990 and 3168 bp) owing to an extra copy of the subrepeat A. The A subrepeats were also shown to be highly homologous to the satellite repeat S12, which is located in large clusters on chromosomes 4, 5, and 6, and is not associated with the rDNA loci. Sequencing of additional S12 clones retrieved from a shotgun genomic library allowed definition of three subfamilies of this repeat based on minor differences in their nucleotide sequences. Two of these subfamilies could be discriminated from the rest of the S12 sequences as well as from the IGS A subrepeats using specific oligonucleotide primers that labeled only a subset of the S12 loci when used in the primed in situ DNA labeling (PRINS) reaction on mitotic chromosomes. These experiments showed that, in spite of the high overall similarity of the IGS A subrepeats and the S12 satellite repeats, there are S12 subfamilies that are divergent from the common consensus and are present at only some of the chromosomes containing the S12 loci. Thus, the subfamilies may have evolved at these loci following the spreading of the A subrepeats from the IGS to genomic regions outside the rDNA clusters.Electronic Supplementary Material Supplementary material is available in the online version of this article at Accession numbers: GenBank AY234364–AY234374. The monomer sequences and additional information about the family of IGS-like repeat S12 will also appear in the PlantSat database (Macas et al. 2002, ) under Accession name Vicia_sativa_IGS-like     

Meiotic chromosome behaviour reflects levels of sequence divergence inSus scrofa domestica satellite DNA

We present a general model for the evolution of chromosome-specific satellite DNA subfamilies.Sus scrofa domestica has a bimodal karyotype with two autosomal subsets of 12 meta-/submetacentric (Mc) and 6 acrocentric (Ac) chromosome types (Mc and Ac subgenomes). We show that the centromeric heterochromatin is characterised by two distinct satellite DNA families designed Mc1 and Ac2. Mc1 is a diverse satellite family of the Mc subgenome of which certain members with a 100 bp repeat unit are found to occur at the pericentromeric regions of each Mc autosome, while others are chromosome-specific, e.g. clone Mc pAv1.5, a higher order repeat variant, which hybridises specifically to chromosome 1. Ac2 is a homogeneous satellite occurring at the subterminal pericentromeric regions of all Ac autosomes. DNA sequence analyses showed that all clones investigated are built up from a 14 bp repeat unit which is highly conserved. In situ hybridisation to meiotic pachytene nuclei revealed a distinct spatial arrangement of the Ac2 centromeric satellite.     

Using a pericentromeric interspersed repeat to recapitulate the phylogeny and expansion of human centromeric segmental duplications

Despite considerable advances in sequencing of the human genome over the past few years, the organization and evolution of human pericentromeric regions have been difficult to resolve. This is due, in part, to the presence of large, complex blocks of duplicated genomic sequence at the boundary between centromeric satellite and unique euchromatic DNA. Here, we report the identification and characterization of an approximately 49-kb repeat sequence that exists in more than 40 copies within the human genome. This repeat is specific to highly duplicated pericentromeric regions with multiple copies distributed in an interspersed fashion among a subset of human chromosomes. Using this interspersed repeat (termed PIR4) as a marker of pericentromeric DNA, we recovered and sequence-tagged 3 Mb of pericentromeric DNA from a variety of human chromosomes as well as nonhuman primate genomes. A global evolutionary reconstruction of the dispersal of PIR4 sequence and analysis of flanking sequence supports a model in which pericentromeric duplications initiated before the separation of the great ape species (>12 MYA). Further, analyses of this duplication and associated flanking duplications narrow the major burst of pericentromeric duplication activity to a time just before the divergence of the African great ape and human species (5 to 7 MYA). These recent duplication exchange events substantially restructured the pericentromeric regions of hominoid chromosomes and created an architecture where large blocks of sequence are shared among nonhomologous chromosomes. This report provides the first global view of the series of historical events that have reshaped human pericentromeric regions over recent evolutionary time.     

Cytological and biochemical characterization of the in situ endonuclease digestion of fixed Tenebrio molitor chromosomes

Cytological and biochemical experiments were undertaken in order to characterize the action of several restriction enzymes on fixed chromosomes of Tenebrio molitor (Coleoptera). EcoRI cuts the satellite DNA of this organism into suunit monomers of 142 bp in naked DNA and acts on fixed chromosomes cleaving and extracting these tandemly repeated sequences present in median centromeric heterochromatin. AluI, in contrast, is unable to attack the satellite sequences but does cut the main band DNA both in naked DNA and in fixed chromosomes. These enzymes therefore permit the in situ localization of satellite DNA or main band DNA in T. molitor. Other enzymes such HinfI or Sau3A do not produce longitudinal differentiation in chromosomes because of the extraction of DNA from satellite and main band DNA regions. In situ hybridization with a satellite DNA probe from T. molitor confirms that the DNA extracted from the chromosomes is the abundant and homogenous highly repeated DNA present in pericentromeric regions. These results plus the analysis of the DNA fractions retained on the slide and solubilized by the action of the restriction enzymes in situ provide evidence that: (a) as an exception to the rule EcoRI (6 bp cutter) is able to produce chromosome banding; (b) the size of the fragments produced by in situ digestion of satellite DNA with EcoRI is not a limiting factor in the extraction; (c) there is a remarkable accord between the action of EcoRI and AluI on naked DNA and on DNA in fixed chromosomes, and (d) the organization of specific chromosome regions seems to be very important in producing longitudinal differentiation on chromosomes.by E.R. Schmidt     

Identification and characterization of functional centromeres of the common bean

In higher eukaryotes, centromeres are typically composed of megabase‐sized arrays of satellite repeats that evolve rapidly and homogenize within a species' genome. Despite the importance of centromeres, our knowledge is limited to a few model species. We conducted a comprehensive analysis of common bean (Phaseolus vulgaris) centromeric satellite DNA using genomic data, fluorescence in situ hybridization (FISH), immunofluorescence and chromatin immunoprecipitation (ChIP). Two unrelated centromere‐specific satellite repeats, CentPv1 and CentPv2, and the common bean centromere‐specific histone H3 (PvCENH3) were identified. FISH showed that CentPv1 and CentPv2 are predominantly located at subsets of eight and three centromeres, respectively. Immunofluorescence‐ and ChIP‐based assays demonstrated the functional significance of CentPv1 and CentPv2 at centromeres. Genomic analysis revealed several interesting features of CentPv1 and CentPv2: (i) CentPv1 is organized into an higher‐order repeat structure, named Nazca, of 528 bp, whereas CentPv2 is composed of tandemly organized monomers; (ii) CentPv1 and CentPv2 have undergone chromosome‐specific homogenization; and (iii) CentPv1 and CentPv2 are not likely to be commingled in the genome. These findings suggest that two distinct sets of centromere sequences have evolved independently within the common bean genome, and provide insight into centromere satellite evolution.     

Analysis of divergence of Alphitobius diaperinus satellite DNA — roles of recombination, replication slippage and gene conversion

Satellite DNA is highly abundant in Alphitobius diaperinus (Tenebrionidae, Coleoptera), comprising 25% of the total genomic DNA. Sequence analysis reveals an average GC content of 50.8% and the presence of three different groups of satellite monomer variants, tH1, tH2 and tH3 with corresponding lengths of 123, 128 and 126 bp. Their mutual homologies range between 65 and 81 %. Sequence comparison shows that the monomer variant tH2 has been formed by a recombination process between tH1 and tH3, which have a low average homology of only 65.15%. The longest stretch of 100% homology between the recombining units is 17 bp and is located 3 to the predicted recombination site. There is also an indication from sequence analysis that replication slippage and gene conversion play a part in the formation of satellite units and contribute to their divergence. The tH1, tH2 and tH3 monomer variants are organized in higher order repeating structures: a dimer, composed of tH1, and tH3, and a trimer containing tH1, tH2 and tH3 in series. The dimeric and trimeric repeat units furthermore create three higher order satellite subfamilies. Two of them contain either tandemly arranged dimers or trimers, while the third one is composed of both types of repeats, mutually interspersed.     

Repertoire and Organization of Human T-Cell Receptor α Region Variable Genes

A contig of YAC, PAC, and cosmid clones that spanned the human T-cell receptor α variable (AV) gene region on chromosome 14 was assembled. PCR primers corresponding to the members of 32 different subfamilies were used to map AV genes on the genomic clones. Nucleotide sequencing of PCR products derived from different genomic clones was used to discriminate between related AV gene segments that coamplified. The presence of individual AV gene segments on genomic clones was further confirmed by hybridization both to clones and to human genomic DNA from several unrelated individuals. These results suggest that the T-cell receptor α (TCRA) region in humans contains at least 46 distinct AV gene segments that can be grouped into 32 subfamilies based on nucleotide homology. Several subfamilies appear to contain additional members detectable by hybridization that do not map to chromosome 14.     

Molecular and cytological characterization of repetitive DNA sequences from the centromeric heterochromatin of <Emphasis Type="Italic">Sciara coprophila</Emphasis>

Sciara coprophila (Diptera, Nematocera) constitutes a classic model to analyze unusual chromosome behavior such as the somatic elimination of paternal X chromosomes, the elimination of the whole paternal, plus non-disjunction of the maternal X chromosome at male meiosis. The molecular organization of the heterochromatin in S. coprophila is mostly unknown except for the ribosomal DNA located in the X chromosome pericentromeric heterochromatin. The characterization of the centromeric regions, thus, is an essential and required step for the establishment of S. coprophila as a model system to study fundamental mechanisms of chromosome segregation. To accomplish such a study, heterochromatic sections of the X chromosome centromeric region from salivary glands polytene chromosomes were microdissected and microcloned. Here, we report the identification and characterization of two tandem repeated DNA sequences from the pericentromeric region of the X chromosome, a pericentromeric RTE element and an AT-rich centromeric satellite. These sequences will be important tools for the cloning of S. coprophila centromeric heterochromatin using libraries of large genomic clones.     

Characterization of the satellite DNA Msat-160 from the species <Emphasis Type="Italic">Chionomys</Emphasis><Emphasis Type="Italic">nivalis</Emphasis> (Rodentia,Arvicolinae)

The satellite DNA Msat-160 has been previously characterized in several species of the genus Microtus. Here we present the characterization of Msat-160 from Chionomys nivalis, a species with a very primitive karyotype. As in other Microtus species analyzed, C. nivalis Msat-160 is AT rich, has a monomer length of 160 bp, is undermethylated and is mainly located in all the pericentromeric heterochromatin of all autosomes and the X chromosome, but is completely absent from the Y chromosome. Hence, our results support the hypothesis that Msat-160 was initially distributed in the pericentromeric heterochromatin of all autosomes and the X chromosome. The taxonomic status of the genus Chionomys in relation to the genus Microtus is a very interesting issue, so we constructed phylogenetic dendrograms using Msat-160 sequences from several Microtus species. Although the results were not informative about this issue, the presence of Msat-160 in C. nivalis and Microtus species suggested that both genera are closely related and that this satellite DNA was present in the common ancestor. Studies of Msat-160 in different arvicoline species could help to determine the origin of this satellite and, perhaps, to establish the phylogenetic relationships of some arvicoline groups.     

Species-specific DNA sequences for identification of somatic hybrids between Lycopersicon esculentum and Solanum acaule

Summary Species-specific highly repeated DNA sequences can be used to screen the progeny of protoplast fusions combining different species. Such probes are easy to clone and can be detected by fast methods, e.g., hybridization to total genomic DNA. Furthermore, due to their high copy number, hybridization signals are strong and represent more than one locus, unlike isozymes or resistance markers. After cloning and screening for species-specific DNA sequences we characterized the highly repeated DNA sequences of the solanaceous species Solanum acaule and Lycopersicon esculentum var. gilva. DNA sequencing and hy ridization revealed a prominent, tandemly arranged satellite DNA repeat of 162 bp in Lycopersicon esculentum and a different satellite repeat of 183 bp, also tandemly organized, in Solanum acaule. Each repeat is absent in the respective other species. Therefore, we have used these DNA repeats as markers to distinguish regenerated interspecific somatic hybrids from the respective fusion partners. These hybrids were clearly identified by Southern hybridization and dot-blot assays to the respective ³²P-labelled satellite DNA.     

Epigenetic profiling of heterochromatic satellite DNA

Sugar beet (Beta vulgaris) chromosomes consist of large heterochromatic blocks in pericentromeric, centromeric, and intercalary regions comprised of two different highly abundant DNA satellite families. To investigate DNA methylation at single base resolution at heterochromatic regions, we applied a method for strand-specific bisulfite sequencing of more than 1,000 satellite monomers followed by statistical analyses. As a result, we uncovered diversity in the distribution of different methylation patterns in both satellite families. Heavily methylated CG and CHG (H=A, T, or C) sites occur more frequently in intercalary heterochromatin, while CHH sites, with the exception of CAA, are only sparsely methylated, in both intercalary and pericentromeric/centromeric heterochromatin. We show that the difference in DNA methylation intensity is correlated to unequal distribution of heterochromatic histone H3 methylation marks. While clusters of H3K9me2 were absent from pericentromeric heterochromatin and restricted only to intercalary heterochromatic regions, H3K9me1 and H3K27me1 were observed in all types of heterochromatin. By sequencing of a small RNA library consisting of 6.76 million small RNAs, we identified small interfering RNAs (siRNAs) of 24 nucleotides in size which originated from both strands of the satellite DNAs. We hypothesize an involvement of these siRNAs in the regulation of DNA and histone methylation for maintaining heterochromatin.     

Satellite DNA in the elm leaf beetle,Xanthogaleruca luteola (Coleoptera,Chrysomelidae): characterization,interpopulation analysis,and chromosome location

In this paper the satellite DNA (stDNA) of the phytophagous beetle Xanthogaleruca luteola is analyzed. It is organized in a tandem repeat of 149-bp-long monomers, has an AT content of 59%, and presents inverted internal repeats. Restriction analysis of the total DNA with methylation-sensitive enzymes suggests that this repetitive DNA is not methylated. Analysis of the electrophoretic mobility of stDNA on non-denaturing polyacrylamide gels showed that this stDNA is not curved. In situ hybridization with a biotinylated probe of the stDNA revealed a pericentromeric localization of these sequences in the majority of the meiotic bivalents. We have studied the stDNA of X. luteola from two populations with very distinct geographical origins. The sequence and phylogenetic analysis of monomers from these two populations showed that the repetitive element is conserved within the species. Putative gene conversion tracts are identified when the different monomers of the same population are compared. These results could indicate the existence of processes of homogenization that would extend these mutations to all the satellite repeats.     

Analysis of divergence of Alphitobius diaperinus satellite DNA — roles of recombination,replication slippage and gene conversion

Satellite DNA is highly abundant in Alphitobius diaperinus (Tenebrionidae, Coleoptera), comprising 25% of the total genomic DNA. Sequence analysis reveals an average GC content of 50.8% and the presence of three different groups of satellite monomer variants, tH1, tH2 and tH3 with corresponding lengths of 123, 128 and 126 bp. Their mutual homologies range between 65 and 81 %. Sequence comparison shows that the monomer variant tH2 has been formed by a recombination process between tH1 and tH3, which have a low average homology of only 65.15%. The longest stretch of 100% homology between the recombining units is 17 bp and is located 3′ to the predicted recombination site. There is also an indication from sequence analysis that replication slippage and gene conversion play a part in the formation of satellite units and contribute to their divergence. The tH1, tH2 and tH3 monomer variants are organized in higher order repeating structures: a dimer, composed of tH1, and tH3, and a trimer containing tH1, tH2 and tH3 in series. The dimeric and trimeric repeat units furthermore create three higher order satellite subfamilies. Two of them contain either tandemly arranged dimers or trimers, while the third one is composed of both types of repeats, mutually interspersed.     

Detection of satellite DNA in Palorus ratzeburgii: Analysis of curvature profiles and comparison with Tenebrio molitor satellite DNA

Very abundant and homogenous satellite DNA has been found in the flour beetle Palorus ratzeburgii, representing 40% of its genome. Sequencing of 14 randomly cloned satelite monomers revealed a conserved monomer length of 142 bp and an average A+T content of 68%. Sequence variation analysis showed that base substitutions, appearing with a frequency of 2.3%, are predominant differences among satellite monomers. The satellite sequence is unique without significant direct repeats and with only two potentially stable inverted repeats. After electrophoresis of satellite monomers on native polyacrylamide gel retarded mobilities characteristic for curved DNA molecules are observed. The curvature profiles and DNA helix axis trajectory are calculated on the basis of three different algorithms. These calculations predict that P ratzeburgii satellite DNA forms a left-handed solenoid superstructure. Comparison of described features with other satellite DNAs reveals some striking similarities with satellite DNA from related species Tenebrio molitor, which belongs to the same family of Tenebrionidae. Both satellites are very abundant and homogenous with the same, highly conserved monomer length, although there is no homology at the nucleotide level. Their monomers, as well as multimers, exhibit very similar retarded electrophoretic mobilities. The calculated curvature profiles predict two bend centers in monomers of each satellite, resulting in a model of left-handed solenoid superstructures of similar appearance.