The use of repetitive DNA in cytogenetic studies of plant sex chromosomes

The structure of sex chromosomes in plants was analyzed by fluorescent in situ hybridization (FISH) with repetitive DNAs. FISH probes were successfully obtained from DNA libraries that were amplified from microdissected sex chromosomes. Some probes hybridized to the subtelomeric regions, where many kinds of repetitive DNAs are located with intrachromosomal similarity of their repeat units rather than interchromosomal similarity. For example, FISH with the subtelomeric repetitive sequence can easily show the location of the pseudoautosomal region (PAR) on the X chromosome of Silene latifolia. The other probes were localized on the interstitial region of the sex chromosomes. The interstitial region contains chloroplast DNAs or neighboring sequences of the internal telomeres, suggesting insertion or translocation occurred during differentiation of the sex chromosome. These data are very informative for understanding the structure of the plant sex chromosomes and their evolutionary process.     

C-banded karyotypes of two Silene species with heteromorphic sex chromosomes.

Mitotic metaphase chromosomes of Silene latifolia (white campion) and Silene dioica (red campion) were studied and no substantial differences between the conventional karyotypes of these two species were detected. The classification of chromosomes into three distinct groups proposed for S. latifolia by Ciupercescu and colleagues was considered and discussed. Additionally, a new small satellite on the shorter arm of homobrachial chromosome 5 was found. Giemsa C-banded chromosomes of the two analysed species show many fixed and polymorphic heterochromatic bands, mainly distally and centromerically located. Our C-banding studies provided an opportunity to better characterize the sex chromosomes and some autosome types, and to detect differences between the two Silene karyotypes. It was shown that S. latifolia possesses a larger amount of polymorphic heterochromatin, especially of the centromeric type. The two Silene sex chromosomes are easily distinguishable not only by length or DNA amount differences but also by their Giemsa C-banding patterns. All Y chromosomes invariably show only one distally located band, and no other fixed or polymorphic bands on this chromosome were observed in either species. The X chromosomes possess two terminally located fixed bands, and some S. latifolia X chromosomes also have an extra-centric segment of variable length. The heterochromatin amount and distribution revealed by our Giemsa C-banding studies provide a clue to the problem of sex chromosome and karyotype evolution in these two closely related dioecious Silene species.     

Structure and variability of human chromosome ends.

Mammalian telomeres are thought to be composed of a tandem array of TTAGGG repeats. To further define the type and arrangement of sequences at the ends of human chromosomes, we developed a direct cloning strategy for telomere-associated DNA. The method involves a telomere enrichment procedure based on the relative lack of restriction endonuclease cutting sites near the ends of human chromosomes. Nineteen (TTAGGG)n-bearing plasmids were isolated, two of which contain additional human sequences proximal to the telomeric repeats. These telomere-flanking sequences detect BAL 31-sensitive loci and thus are located close to chromosome ends. One of the flanking regions is part of a subtelomeric repeat that is present at 10 to 25% of the chromosome ends in the human genome. This sequence is not conserved in rodent DNA and therefore should be a helpful tool for physical characterization of human chromosomes in human-rodent hybrid cell lines; some of the chromosomes that may be analyzed in this manner have been identified, i.e., 7, 16, 17, and 21. The minimal size of the subtelomeric repeat is 4 kilobases (kb); it shows a high frequency of restriction fragment length polymorphisms and undergoes extensive de novo methylation in somatic cells. Distal to the subtelomeric repeat, the chromosomes terminate in a long region (up to 14 kb) that may be entirely composed of TTAGGG repeats. This terminal segment is unusually variable. Although sperm telomeres are 10 to 14 kb long, telomeres in somatic cells are several kilobase pairs shorter and very heterogeneous in length. Additional telomere reduction occurs in primary tumors, indicating that somatic telomeres are unstable and may continuously lose sequences from their termini.     

'Junk' DNA and phenotypic evolution in Silene section Siphonomorpha.

One of the long-standing mysteries in genomic evolution is the observation that much of the genome is composed of repetitive DNA, resulting in inter- and intraspecific variation in nuclear DNA content. Our discovery of a negative correlation between nuclear DNA content and flower size in Silene latifolia has been supported by our subsequent investigation of changes in DNA content as a correlated response to selection on flower size. Moreover, we have observed a similar trend across a range of related dioecious species in Silene sect. Elisanthe. Given the presence of sex chromosomes in dioecious Silene species, and the tendency of sex chromosomes to accumulate repetitive DNA, it seems plausible that dioecious species undergo genomic evolution in ways that differ from what one might expect in hermaphroditic species. Specifically, we query whether the observed relationship between nuclear DNA content and flower size observed in dioecious Silene is a peculiarity of sex chromosome evolution. In the present study we investigated nuclear DNA content and flower size variation in hermaphroditic species of Silene sect. Siphonomorpha, as close relatives of the dioecious species studied previously. Although the nuclear DNA contents of these species were lower than those for species in sect. Elisanthe, there was still significant intra- as well as interspecific variation in nuclear DNA content. Flower size variation was found among species of sect. Siphonomorpha for petal claw and petal limb lengths, but not for calyx diameter. This last trait varies extensively in sect. Elisanthe, in part due to sex-specific selection. A negative correlation with nuclear DNA content was found across populations for petal limb length, but not for other floral dimensions. We conclude that impacts of nuclear DNA content on phenotypic evolution do manifest themselves in hermaphroditic species, so that the effects observed in sect. Elisanthe, and particularly in S. latifolia, while perhaps amplified by the genomic impacts of sex chromosomes, are not limited to dioecious taxa.     

Characterization of telomere-subtelomere junctions in <Emphasis Type="Italic"> Silene latifolia</Emphasis>

Telomere-associated regions represent boundaries between the relatively homogeneous telomeres and the subtelomeres, which show much greater heterogeneity in chromatin structure and DNA composition. Although a major fraction of subtelomeres is usually formed by a limited number of highly repeated DNA sequence families, their mutual arrangement, attachment to telomeres and the presence of interspersed unique or low-copy-number sequences make these terminal domains chromosome specific. In this study, we describe the structures of junctions between telomeres and a major subtelomeric repeat of the plant Silene latifolia, X43.1. Our results show that on individual chromosome arms, X43.1 is attached to the telomere either directly at sites corresponding to nucleosome boundaries previously mapped in this sequence, or via other spacer sequences, both previously characterized and newly described ones. Sites of telomere junctions are non-random in all the telomere-associated sequences analysed. These data obtained at the molecular level have been verified using in situ hybridization to metaphase chromosomes and extended DNA fibres.     

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.     

The clustering of four subfamilies of satellite DNA at individual chromosome ends in Silene latifolia.

The satellite DNA (satDNA) on the ends of chromosomes has been isolated and characterized in the dioecious plant Silene latifolia. BAC clones containing large numbers of repeat units of satDNA in a tandem array were isolated to examine the clustering of the repeat units. satDNA repeat units were purified from each isolated BAC clone and sequenced. To investigate pairwise similarities among the repeat units, a phylogenetic tree was constructed using the neighbor-joining algorithm. The repeat units derived from 7 BAC clones were grouped into SacI, KpnI, #11F02, and #16E07 subfamilies. The SacI and KpnI subfamilies have been reported previously. Multicolored fluorescence in situ hybridization (FISH) using SacI or KpnI subfamily probes resulted in different signal intensities and locations at the chromosomal ends, indicating that each chromosomal end has a unique composition of subfamilies of satDNA. For example, the p arm of the X chromosome exhibited signal composition similar to that on the pseudo autosomal region (PAR) of the Y chromosome, but not to that on the q arm of the X chromosome. The satDNA has not been completely homogenized in the S. latifolia genome. Each subfamily is available for a probe of FISH karyotyping.     

The dynamic nature and evolutionary history of subtelomeric and pericentromeric regions

The organization and evolution of the subtelomeric and pericentromeric regions of human chromosomes exhibit unique characteristics compared to other regions of the genome. As shown in Fig. 1 the functional elements of the centromere and telomere are comprised of highly repetitive DNA sequences, which are responsible for carrying out the main mechanistic duties of these two regions: chromosome segregation and end replication, respectively. The nature of the repeats in these two regions and their function have been reviewed separately and, therefore, will not be discussed in more detail here (Sullivan et al., 1996, 2001; McEachern et al., 2000; Henikoff et al., 2001). Adjacent to these functional element regions, the centromere and telomere regions share an interesting architecture as depicted in Fig. 1. For both pericentromeric and subtelomeric regions, blocks of recent genomic duplications form a zone of shared sequence homologies between certain subsets of human chromosomes. The dynamic nature and evolutionary history of these regions and the unique DNA sequence adjacent to them will be the focus of this review.     

Semi-automatic laser beam microdissection of the Y chromosome and analysis of Y chromosome DNA in a dioecious plant, Silene latifolia

Silene latifolia has heteromorphic sex chromosomes, the X and Y chromosomes. The Y chromosome, which is thought to carry the male determining gene, was isolated by UV laser microdissection and amplified by degenerate oligonucleotide-primed PCR. In situ chromosome suppression of the amplified Y chromosome DNA in the presence of female genomic DNA as a competitor showed that the microdissected Y chromosome DNA did not specifically hybridize to the Y chromosome, but hybridized to all chromosomes. This result suggests that the Y chromosome does not contain Y chromosome-enriched repetitive sequences. A repetitive sequence in the microdissected Y chromosome, RMY1, was isolated while screening repetitive sequences in the amplified Y chromosome. Part of the nucleotide sequence shared a similarity to that of X-43.1, which was isolated from microdissected X chromosomes. Since fluorescence in situ hybridization analysis with RMY1 demonstrated that RMY1 was localized at the ends of the chromosome, RMY1 may be a subtelomeric repetitive sequence. Regarding the sex chromosomes, RMY1 was detected at both ends of the X chromosome and at one end near the pseudoautosomal region of the Y chromosome. The different localization of RMY1 on the sex chromosomes provides a clue to the problem of how the sex chromosomes arose from autosomes.     

The genome of Oscheius tipulae: determination of size, complexity, and structure by DNA reassociation using fluorescent dye.

This work describes the physicochemical characterization of the genome and telomere structure from the nematode Oscheius tipulae CEW1. Oscheius tipulae is a free-living nematode belonging to the family Rhabditidae and has been used as a model system for comparative genetic studies. A new protocol that combines fluorescent detection of double-stranded DNA and S1 nuclease was used to determine the genome size of O. tipulae as 100.8 Mb (approximately 0.1 pg DNA/haploid nucleus). The genome of this nematode is made up of 83.4% unique copy sequences, 9.4% intermediate repetitive sequences, and 7.2% highly repetitive sequences, suggesting that its structure is similar to those of other nematodes of the genus Caenorhabditis. We also showed that O. tipulae has the same telomere repeats already found in Caenorhabditis elegans at the ends and in internal regions of the chromosomes. Using a cassette-ligation-mediated PCR protocol we were able to obtain 5 different putative subtelomeric sequences of O. tipulae, which show no similarity to C. elegans or C. briggsae subtelomeric regions. DAPI staining of hermaphrodite gonad cells show that, as detected in C. elegans and other rhabditids, O. tipulae have a haploid complement of 6 chromosomes.     

Characterization of ancestral chromosome fusion points in the Indian muntjac deer

Tandem fusion, a rare evolutionary chromosome rearrangement, has occurred extensively in muntjac karyotypic evolution, leading to an extreme fusion karyotype of 6/7 (female/male) chromosomes in the Indian muntjac. These fusion chromosomes contain numerous ancestral chromosomal break and fusion points. Here, we designed a composite polymerase chain reaction (PCR) strategy which recovered DNA fragments that contained telomere and muntjac satellite DNA sequence repeats. Nested PCR confirmed the specificity of the products. Two-color fluorescence in situ hybridization (FISH) with the repetitive sequences obtained and T₂AG₃ telomere probes showed co-localization of satellite and telomere sequences in Indian muntjac chromosomes. Adjacent telomere and muntjac satellite sequences were also seen by fiber FISH. These data lend support to the involvement of telomere and GC-rich satellite DNA sequences during muntjac chromosome fusions.Communicated by E.A. NiggAccession numbers: AY322158, AY322159, AY322160     

Distribution of interstitial telomere-like repeats and their adjacent sequences in a dioecious plant, <Emphasis Type="Italic">Silene latifolia</Emphasis>

The dioecious plant Silene latifolia has large, heteromorphic X and Y sex chromosomes that are thought to be derived from rearrangements of autosomes. To reveal the origin of the sex chromosomes in S. latifolia, we isolated and characterized telomere-homologous sequences from intra-chromosomal regions (interstitial telomere-like repeats; ITRs) and ITR-adjacent sequences (IASs). Nine genomic DNA fragments with degenerate 84- to 175-bp ITRs were isolated from a genomic library and total genome of male plants. Comparing the nucleotide sequences, the IASs of the nine ITRs were classified into seven elements (IAS-a, IAS-b, IAS-c, IAS-d, IAS-e, IAS-f, and IAS-g) by sequence similarity. The ITRs were grouped into two classes (class-I and -II ITRs) according to the classification of IASs. The class-I ITRs were sub-grouped into three subclasses (subclasses-IA, -IB, and -IC ITRs) based on the arrangement of IAS elements. By contrast, the class-II ITR was located between two different IASs (IAS-f and IAS-g). Genomic Southern analyses showed that both the male and female genomes contained six (IAS-f) to 153 (IAS-d) copies of each IAS per haploid genome. Fluorescence in situ hybridization analyses showed that one IAS element, IAS-d, was distributed in the interstitial and proximal regions of the sex chromosomes of S. latifolia. The distribution of IAS-d is important evidence for past telomere-mediated chromosome rearrangements during the evolution of the sex chromosomes of S. latifolia.     

Sequence and sequence variation within the 1.688 g/cm3 satellite DNA of Drosophila melanogaster.

We have determined the complete nucleotide sequence of the monomer repeating unit of the 1.688 g/cm³ satellite DNA from Drosophila melanogaster. This satellite DNA, which makes up 4% of the Drosophila genome and is located primarily on the sex chromosomes, has a repeat unit 359 base-pairs in length. This complex sequence is unrelated to the other three major satellite DNAs present in this species, each of which contains a very short repeated sequence only 5 to 10 base-pairs long. The repeated sequence is more similar to the complex repeating units found in satellites of mammalian origin in that it contains runs of adenylate and thymidylate residues. We have determined the nature of the sequence variations in this DNA by restriction nuclease cleavage and by direct sequence determination of (1) individual monomer units cloned in hybrid plasmids, (2) mixtures of adjacent monomers from a cloned segment of this satellite DNA, (3) mixtures of monomer units isolated by restriction nuclease cleavage of total 1.688 g/cm³ satellite DNA. Both direct sequence determination and restriction nuclease cleavage indicate that certain positions in the repeat can be highly variable with up to 50% of certain restriction sites having altered recognition sequences. Despite the high degree of variation at certain sites, most positions in the sequence are highly conserved. Sequence analysis of a mixture of 15 adjacent monomer units detected only nine variable positions out of 359 base-pairs. Total satellite DNA showed only four additional positions. While some variability would have been missed due to the sequencing methods used, we conclude that the variation from one repeat to the next is not random and that most of the satellite repeat is conserved. This conservation may reflect functional aspects of the repeated DNA, since we have shown earlier that part of this sequence serves as a binding site for a sequence-specific DNA binding protein isolated from Drosophila embryos (Hsieh &; Brutlag, 1979).     

Genetics of sex determination in flowering plants

Most flowering plant species are hermaphroditic, but a small number of species in most plant families are unisexual (i.e., an individ-ual will produce only male or female gametes). Because species with unisexual flowers have evolved repeatedly from hermaphroditic progenitors, the mechanisms controlling sex determination in flowering plants are extremely diverse. Sex is most strongly determined by genotype in all species but the mechanisms range from a single controlling locus to sex chromosomes bearing several linked locirequired for sex determination. Plant hormones also influence sex expression with variable effects from species to species. Here, we review the genetic control of sex determination from a number of plant species to illustrate the variety of extant mechanisms. We emphasize species that are now used as models to investigate the molecular biology of sex determination. We also present our own investigations of the structure of plant sex chromosomes of white campion (Silene latifolia - Melan-drium album). The cytogenetic basis of sex determination in white campion is similar to mammals in that it has a male-specific Y-chromosome that carries dominant male determining genes. If one copy of this chromosome is in the genome, the plant is male. Otherwise it is female. Like mammalian Y-chromosomes, the white campion Y-chromosome is rich in repetitive DNA. We isolated repetitive sequences from microdissected Y-chromosomes of white campion to study the distribution of homologous repeated sequences on the Y-chromosome and the other chromosomes. We found the Y to be especially rich in repetitive sequences that were generally dispersed over all the white campion chromosomes. Despite its repetitive character, the Y-chromosome is mainly euchromatic. This may be due to the relatively recent evolution of the white campion sex chromosomes compared to the sex chromosomes of animals. © 1994 Wiley-Liss, Inc.     

Satellite DNA from the Y chromosome of the malaria vector Anopheles gambiae

Satellite DNA is an enigmatic component of genomic DNA with unclear function that has been regarded as "junk." Yet, persistence of these tandem highly repetitive sequences in heterochromatic regions of most eukaryotic chromosomes attests to their importance in the genome. We explored the Anopheles gambiae genome for the presence of satellite repeats and identified 12 novel satellite DNA families. Certain families were found in close juxtaposition within the genome. Six satellites, falling into two evolutionarily linked groups, were investigated in detail. Four of them were experimentally confirmed to be linked to the Y chromosome, whereas their relatives occupy centromeric regions of either the X chromosome or the autosomes. A complex evolutionary pattern was revealed among the AgY477-like satellites, suggesting their rapid turnover in the A. gambiae complex and, potentially, recombination between sex chromosomes. The substitution pattern suggested rolling circle replication as an array expansion mechanism in the Y-linked 53-bp satellite families. Despite residing in different portions of the genome, the 53-bp satellites share the same monomer lengths, apparently maintained by molecular drive or structural constraints. Potential functional centromeric DNA structures, consisting of twofold dyad symmetries flanked by a common sequence motif, have been identified in both satellite groups.     

Two extended arrays of a satellite DNA sequence at the centromere and at the short-arm telomere of Chinese hamster chromosome 5

We have cloned a Chinese hamster chromosome-specific repeated sequence (SatCH5). This satellite is composed of a 33-bp unit organized in two extended tandem arrays. It is localized at the centromere and at the short-arm subtelomere of chromosome 5. Altogether, SatCH5 covers about 1-2 Mb per diploid genome and is not present in other species, including the Syrian hamster and mouse. Since it is known in the Chinese hamster and numerous other vertebrate species that telomeric (TTAGGG)n repeats are localized at the centromeres of several chromosomes, we studied the localization of SatCH5 relative to (TTAGGG)n sequences. Using two-color fluorescence in situ hybridization on stretched chromosomes and on DNA fibers, we have shown that at the centromere of chromosome 5 SatCH5 and the (TTAGGG)n arrays are contiguous. SatCH5 is the first chromosome-specific repetitive sequence located at both the pericentromeric and subtelomeric regions of the same chromosome.     

DNA, chromosomes, and in situ hybridization.

In situ hybridization is a powerful and unique technique that correlates molecular information of a DNA sequence with its physical location along chromosomes and genomes. It thus provides valuable information about physical map position of sequences and often is the only means to determine abundance and distribution of repetitive sequences making up the majority of most genomes. Repeated DNA sequences, composed of units of a few to a thousand base pairs in size, occur in blocks (tandem or satellite repeats) or are dispersed (including transposable elements) throughout the genome. They are often the most variable components of a genome, often being species and, occasionally, chromosome specific. Their variability arises through amplification, diversification and dispersion, as well as homogenization and loss; there is a remarkable correlation of molecular sequence features with chromosomal organization including the length of repeat units, their higher order structures, chromosomal locations, and dispersion mechanisms. Our understanding of the structure, function, organization, and evolution of genomes and their evolving repetitive components enabled many new cytogenetic applications to both medicine and agriculture, particularly in diagnosis and plant breeding.     

PlantSat: a specialized database for plant satellite repeats

MOTIVATION: Tandemly organized repetitive sequences (satellite DNA) are widespread in complex eukaryotic genomes. In plants, satellite repeats often represent a substantial part of nuclear DNA but only a little is known about the molecular mechanisms of their amplification and their possible role(s) in genome evolution and function. Unfortunately, addressing these questions via characterization of general sequence properties of known satellite repeats has been hindered by a difficulty in obtaining a complete and unbiased set of sequence data for this analysis. This is mainly due to the presence of multiple entries of homologous sequences and of single entries that contain more than one repeated unit (monomer) in the public databases. RESULTS: We have established a computer database specialized for plant satellite repeats (PlantSat) that integrates sequence data available from various resources with supplementary information including repeat consensus sequences, abundances, and chromosomal localizations. The sequences are stored as individual repeat monomers grouped into families, which simplifies their computer analysis and makes it more accurate. Using this feature, we have performed a basic sequence analysis of the whole set of plant satellite repeats with respect to their monomer length and nucleotide composition. The analysis revealed several preferred length ranges of the monomers (approximately 165 bp and its multiples) and an over-representation of the AA/TT dinucleotide in the repeats. We have also detected an enrichment of satellite DNA sequences for the motif CAAAA that is supposed to be involved in breakage-reunion of repeated sequences.     

Rapid divergence of repetitive DNAs in Brassica relatives

Centromeric, subtelomeric, and telomeric repetitive DNAs were characterized in Brassica species and the related Raphanus sativus and Arabidopsis thaliana. In general, rapid divergence of the repeats was found. The centromeric tandem satellite repeats were differentially distributed in the species studied, suggesting that centromeric repeats have diverged during the evolution of the A/C and B genome lineages. Sequence analysis of centromeric repeats suggested rapid evolution. Pericentromere-associated retrotransposons were identified and showed divergence during the evolution of the lineages as centromeric repeats. A novel subtelomeric tandem repeat from B. nigra was found to be conserved across the diploid Brassica genomes; however, this sequence was not identified in the related species. In contrast to previous studies, interstitial telomere-like repeats were identified in the pericentromeres of Brassica chromosomes, and these repeats may be associated with genomic stability. These results provide insight into genome evolution during polyploidization in Brassica and divergence within the Brassicaceae.     

Characterization of Interrelated Sequence Motifs in Four Satellite DNAs and Their Distribution in the Genome of the Mollusc Donax trunculus

A family of four satellite DNAs has been characterized in the genome of the bivalve mollusc, Donax trunculus. All share HindIII sites, a similar monomer length of about 160 base pairs (bp), and the related oligonucleotide motifs GGTCA and GGGTTA, repeated six to 15 times within the repetitive units. The motif GGTCA is common to all members of the satellite family. It is present in three of them in both orientations, interspersed within nonrepetitive DNA sequences. The hexanucleotide GGGTTA appears to be the main building element of one of the satellites forming a prominent subrepeat structure in conjunction with the 5-bp motif. The former has been also found in perfect tandem repeats in a junction region adjacent to the proper satellite sequence. Southern analysis has revealed that (GGGTTA)_n and/or related sequences are abundant and widely distributed in the D. trunculus genome. The distribution observed is consistent with the concurrence of the scattering of short sequence motifs throughout the genome and the spread of longer DNA segments, with concomitant formation of satellite monomer repeats. Both kinds of dispersion may have contributed to the observed complex arrangement of the HindIII satellite DNA family in Donax. Received: 28 May 1996 / Accepted: 30 July 1996