Genome and chromosome structure: Twelve dynamic and evolving genomes

Chromosomes are not inert structures that haul the genome through cell division. The dynamic properties of chromosomes, during the cell cycle, the lifetime of the organism and across evolutionary time, featured prominently at the 49(th) Annual Drosophila Research Conference. Platform presentations, workshops and posters focused on many aspects of chromosome structure and function including chromosome interactions such as trans-silencing and pairing between homologous and non-homologous chromosomes, specialized portions of the chromosome including the centromere and telomeres, the structure, function and evolution of the large heterochromatic domains such as the Y and 4(th) chromosomes, centric heterochromatin and subtelomeric heterochromatin. The speed of evolutionary changes in these regions, and the consequences for speciation and hybrid-incompatibility, were recurring themes. Finally, there was considerable new insight offered into the mechanics by which chromosomes are rearranged and changes in the types of alterations occurring over the lifetime of the organism, which can result in novel genes and gene flow between chromosomes. The availability of the twelve sequenced Drosophila genomes has allowed new insights into the structure, function and evolutionary transformation of chromosomes and genomes that will continue to transform our view of the chromosome as a dynamic and flexible entity that houses and regulates the genome.     

Characterization and comparative analysis of DNA from the pericentric heterochromatin of chromosome 2 of Anopheles atroparvus V. Tiel (Culicidae, Diptera)

The minilibrary containing DNA sequences from the diffuse pericentric heterochromatin from the right arm of Anopheles atroparvus V. Tiel (Culicidae, Diptera) chromosome 2 (2R) was generated by use of chromosome microdissection technique. Southern-blot hybridization of the minilibrary fragments with the labeled genomic DNA of A. atroparvus and analysis of their primary structure showed that this heterochromatin region contained repeated DNA sequences differed by their primary structure and the number of copies. These were mostly AT-rich sequences harboring the features characteristic of the S/MAR regions. Based on the clones homology to the sequences from the An. gambiae and Drosophila melanogaster genomes, it was demonstrated that the pericentric heterochromatin from the right arm of An. atroparvus chromosome 2 contained gypsy-like transposable elements, as well as the sequences homologous to the structural genes. In situ hybridization with the chromosomes of A. atroparvus and of the two representatives of the Anopheles maculipennis species complex, A. messeae and A. beklemishevi, showed that pericentric regions of all these chromosomes contained DNA sequences homologous to the sequences from the region-specific minilibrary. Cloned fragments of conserved repetitive DNA revealed upon interspecific Southern-blot hybridization of the clones with the labeled genomic DNA of A. messeae can be utilized in further investigations of evolutionary rearrangements of the pericentric heterochromatin within the Anopheles maculipennis species complex.     

Simple Quantitative PCR Approach to Reveal Naturally Occurring and Mutation-Induced Repetitive Sequence Variation on the Drosophila Y Chromosome

Heterochromatin is a significant component of the human genome and the genomes of most model organisms. Although heterochromatin is thought to be largely non-coding, it is clear that it plays an important role in chromosome structure and gene regulation. Despite a growing awareness of its functional significance, the repetitive sequences underlying some heterochromatin remain relatively uncharacterized. We have developed a real-time quantitative PCR-based method for quantifying simple repetitive satellite sequences and have used this technique to characterize the heterochromatic Y chromosome of Drosophila melanogaster. In this report, we validate the approach, identify previously unknown satellite sequence copy number polymorphisms in Y chromosomes from different geographic sources, and show that a defect in heterochromatin formation can induce similar copy number polymorphisms in a laboratory strain. These findings provide a simple method to investigate the dynamic nature of repetitive sequences and characterize conditions which might give rise to long-lasting alterations in DNA sequence.     

Order and disorder in bacterial genomes

The availability of sequenced bacterial genomes allows a deeper understanding of their organizational features that are related with fundamental cellular processes such as coordinated gene expression, chromosome replication and cell division. Nevertheless, recent genome comparisons and experimental work highlighted the fluidity of bacterial chromosomes, including genome rearrangements that imperil the selective features of chromosome order. As a result, the clash between elements generating rearrangements and chromosome organization is a classic case of evolutionary conflict.     

Polymorphism of heterochromatin C-blocks of rye genome chromosomes in rye-wheat amphidiploids and their chromosome substitution lines

Polymorphism of heterochromatin C-blocks in chromosomes of rye genome has been studied in the F1BC1 hybrids and the D/A substitution lines of rye-wheat amphidiploids (Verasen x L374, Novosibirskaya x x L246)--secalotriticum for revealing cytogenetic markers of rye chromosomes. An increase in polymorphism for the presence and value of heterochromatin blocks in chromosomes of rye genome was shown in the F1BC1 hybrids ((Verasen x L374) x L145 x L145) and unstable genotypes of the F6BC1 chromosome substitution lines (line 118 (Novosibirskaya x L246) x Reso x Reso), line 104 ((Verasen x x L374) x Garmoniya x Garmoniya) that was related to activation of mobile genetic elements present in cereals. Heterochromatin markers of all seven chromosomes in rye genome were revealed. The polymorphism system of heterochromatin blocks may serve as a marker for specificity of the linear chromosome structure during reconstruction of synthetic cereal genomes, and as a test for cytological and morphogenetic stability of hybrid polygenomes in a series of generations.     

Analysis of Drosophila species genome size and satellite DNA content reveals significant differences among strains as well as between species

The size of eukaryotic genomes can vary by several orders of magnitude, yet genome size does not correlate with the number of genes nor with the size or complexity of the organism. Although "whole"-genome sequences, such as those now available for 12 Drosophila species, provide information about euchromatic DNA content, they cannot give an accurate estimate of genome sizes that include heterochromatin or repetitive DNA content. Moreover, genome sequences typically represent only one strain or isolate of a single species that does not reflect intraspecies variation. To more accurately estimate whole-genome DNA content and compare these estimates to newly assembled genomes, we used flow cytometry to measure the 2C genome values, relative to Drosophila melanogaster. We estimated genome sizes for the 12 sequenced Drosophila species as well as 91 different strains of 38 species of Drosophilidae. Significant differences in intra- and interspecific 2C genome values exist within the Drosophilidae. Furthermore, by measuring polyploid 16C ovarian follicle cell underreplication we estimated the amount of satellite DNA in each of these species. We found a strong correlation between genome size and amount of satellite underreplication. Addition and loss of heterochromatin satellite repeat elements appear to have made major contributions to the large differences in genome size observed in the Drosophilidae.     

The Epigenome of Evolving Drosophila Neo-Sex Chromosomes: Dosage Compensation and Heterochromatin Formation

Sex chromosomes originated from autosomes but have evolved a highly specialized chromatin structure. Drosophila Y chromosomes are composed entirely of silent heterochromatin, while male X chromosomes have highly accessible chromatin and are hypertranscribed as a result of dosage compensation. Here, we dissect the molecular mechanisms and functional pressures driving heterochromatin formation and dosage compensation of the recently formed neo-sex chromosomes of Drosophila miranda. We show that the onset of heterochromatin formation on the neo-Y is triggered by an accumulation of repetitive DNA. The neo-X has evolved partial dosage compensation and we find that diverse mutational paths have been utilized to establish several dozen novel binding consensus motifs for the dosage compensation complex on the neo-X, including simple point mutations at pre-binding sites, insertion and deletion mutations, microsatellite expansions, or tandem amplification of weak binding sites. Spreading of these silencing or activating chromatin modifications to adjacent regions results in massive mis-expression of neo-sex linked genes, and little correspondence between functionality of genes and their silencing on the neo-Y or dosage compensation on the neo-X. Intriguingly, the genomic regions being targeted by the dosage compensation complex on the neo-X and those becoming heterochromatic on the neo-Y show little overlap, possibly reflecting different propensities along the ancestral chromosome that formed the sex chromosome to adopt active or repressive chromatin configurations. Our findings have broad implications for current models of sex chromosome evolution, and demonstrate how mechanistic constraints can limit evolutionary adaptations. Our study also highlights how evolution can follow predictable genetic trajectories, by repeatedly acquiring the same 21-bp consensus motif for recruitment of the dosage compensation complex, yet utilizing a diverse array of random mutational changes to attain the same phenotypic outcome.     

Similarities in the chromosomal distribution of AG and AC repeats within and between Drosophila, human and barley chromosomes

Two simple sequence repeats (SSRs), AG and AC, were mapped directly in the metaphase chromosomes of man and barley (Hordeum vulgare L.), and in the metaphase and polytene chromosomes of Drosophila melanogaster. To this end, synthetic oligonucleotides corresponding to (AG)(12) and (AC)(8) were labelled by the random primer technique and used as probes in fluorescent in situ hybridisation (FISH) under high stringency and strict washing conditions. The distribution and intensity of the signals for the repeat sequences were found to be characteristic of the chromosomes and genomes of the three species analysed. The AC repeat sites were uniformly dispersed along the euchromatic segments of all three genomes; in fact, they were largely excluded from the heterochromatin. The Drosophila genome showed a high density of AC sequences on the X chromosome in both mitotic and polytene nuclei. In contrast, the AG repeats were associated with the euchromatic regions of the polytene chromosomes (and in high density on the X chromosome), but were only seen in specific heterochromatic regions in the mitotic chromosomes of all three species. In Drosophila, the AG repeats were exclusively distributed on the tips of the Y chromosome and near the centromere on both arms of chromosome 2. In barley and man, AG repeats were associated with the centromeres (of all chromosomes) and nucleolar organizer regions, respectively. The conserved chromosome distribution of AC within and between these three phylogenetically distant species, and the association of AG in specific chromosome regions with structural or functional properties, suggests that long clusters of these repeats may have some, as yet unknown, role.     

Constitutive heterochromatin polymorphisms in human chromosomes identified by whole comparative genomic hybridization

Whole comparative genomic hybridization (W-CGH) is a new technique that reveals cryptic differences in highly repetitive DNA sequences, when different genomes are compared using metaphase or interphase chromosomes. W-CGH provides a quick approach to identify differential expansion of these DNA sequences at the single-chromosome level in the whole genome. In this study, we have determined the frequency of constitutive chromatin polymorphisms in the centromeric regions of human chromosomes using a whole-genome in situ cross-hybridization method to compare the whole genome of five different unrelated individuals. Results showed that the pericentromeric constitutive heterochromatin of chromosome 6 exhibited a high incidence of polymorphisms in repetitive DNA families located in pericentromeric regions. The constitutive heterochromatin of chromosomes 5 and 9 was also identified as highly polymorphic. Although further studies are necessary to corroborate and assess the overall incidence of these polymorphisms in human populations, the use of W-CGH could be pertinent and of clinical relevance to assess rapidly, from a chromosomal viewpoint, genome similarities and differences in closely related genomes such as those of relatives, or in more specific situations such as bone marrow transplantation where chimerism is produced in the recipient.     

Cytogenetic comparison of N-genome Aegilops L. species

Differential C-banding and in situ hybridization were employed in a cytogenetic comparison of thee N-genome Aegilops species: diploid Ae. uniaristata, tetraploid Ae. ventricosa, and hexaploid Ae. recta. The formation of Ae. recta was shown to involve only minor functional modifications of the parental genomes, while intraspecific divergence was accompanied by large genome rearrangements, namely, translocations involving the total chromosome arms of all of the three genomes. The formation of tetraploid Ae. ventricosa involved substantial structural chromosome rearrangements, including a partial deletion of the short arm of chromosome 5D, including the nucleolus-organizing region; a redistribution of C bands on chromosomes of the D and N genomes along with a reduction of the heterochromatin content; and a considerable decrease in the hybridization intensity of the pAs1 repeat. Chromosomes of the Ae. ventricosa D genome were more similar to chromosomes of the Ae. crassa D1 genome than to Ae. tauschii chromosomes.     

Germ line imprinting in Drosophila: Epigenetics in search of function

Germ line imprinting produces parent-specific differences in the behavior of chromosomes or expression of genes. Epigenetic marks, placed on chromosomes in the parental germ line, govern classical imprinted effects such as chromosomal inactivation, chromosome elimination and mono-allelic expression. Germ line imprinting occurs in insects, plants and mammals. Several Drosophila systems display imprinted effects. In spite of this, many aspects of imprinting in flies, including the normal function of this process, remain mysterious. Transgenerational inheritance of epigenetic marks is a powerful force in genome regulation. Elucidation of the mechanism of imprint establishment and maintenance in a model organism, such as Drosophila, is thus of great interest. In this review we summarize the primary systems that have been used to study imprinting in flies and speculate on the origin and biological function of imprinting in Drosophila.     

A new genomic evolutionary model for rearrangements, duplications, and losses that applies across eukaryotes and prokaryotes

Genomic rearrangements have been studied since the beginnings of modern genetics and models for such rearrangements have been the subject of many papers over the last 10 years. However, none of the extant models can predict the evolution of genomic organization into circular unichromosomal genomes (as in most prokaryotes) and linear multichromosomal genomes (as in most eukaryotes). Very few of these models support gene duplications and losses--yet these events may be more common in evolutionary history than rearrangements and themselves cause apparent rearrangements. We propose a new evolutionary model that integrates gene duplications and losses with genome rearrangements and that leads to genomes with either one (or a very few) circular chromosome or a collection of linear chromosomes. Our model is based on existing rearrangement models and inherits their linear-time algorithms for pairwise distance computation (for rearrangement only). Moreover, our model predictions fit observations about the evolution of gene family sizes and agree with the existing predictions about the growth in the number of chromosomes in eukaryotic genomes.     

Chromosomes in the flow to simplify genome analysis

Nuclear genomes of human, animals, and plants are organized into subunits called chromosomes. When isolated into aqueous suspension, mitotic chromosomes can be classified using flow cytometry according to light scatter and fluorescence parameters. Chromosomes of interest can be purified by flow sorting if they can be resolved from other chromosomes in a karyotype. The analysis and sorting are carried out at rates of 10(2)-10(4) chromosomes per second, and for complex genomes such as wheat the flow sorting technology has been ground-breaking in reducing genome complexity for genome sequencing. The high sample rate provides an attractive approach for karyotype analysis (flow karyotyping) and the purification of chromosomes in large numbers. In characterizing the chromosome complement of an organism, the high number that can be studied using flow cytometry allows for a statistically accurate analysis. Chromosome sorting plays a particularly important role in the analysis of nuclear genome structure and the analysis of particular and aberrant chromosomes. Other attractive but not well-explored features include the analysis of chromosomal proteins, chromosome ultrastructure, and high-resolution mapping using FISH. Recent results demonstrate that chromosome flow sorting can be coupled seamlessly with DNA array and next-generation sequencing technologies for high-throughput analyses. The main advantages are targeting the analysis to a genome region of interest and a significant reduction in sample complexity. As flow sorters can also sort single copies of chromosomes, shotgun sequencing DNA amplified from them enables the production of haplotype-resolved genome sequences. This review explains the principles of flow cytometric chromosome analysis and sorting (flow cytogenetics), discusses the major uses of this technology in genome analysis, and outlines future directions.     

The condensin I subunit Barren/CAP-H is essential for the structural integrity of centromeric heterochromatin during mitosis

During cell division, chromatin undergoes structural changes essential to ensure faithful segregation of the genome. Condensins, abundant components of mitotic chromosomes, are known to form two different complexes, condensins I and II. To further examine the role of condensin I in chromosome structure and in particular in centromere organization, we depleted from S2 cells the Drosophila CAP-H homologue Barren, a subunit exclusively associated with condensin I. In the absence of Barren/CAP-H the condensin core subunits DmSMC4/2 still associate with chromatin, while the other condensin I non-structural maintenance of chromosomes family proteins do not. Immunofluorescence and in vivo analysis of Barren/CAP-H-depleted cells showed that mitotic chromosomes are able to condense but fail to resolve sister chromatids. Additionally, Barren/CAP-H-depleted cells show chromosome congression defects that do not appear to be due to abnormal kinetochore-microtubule interaction. Instead, the centromeric and pericentromeric heterochromatin of Barren/CAP-H-depleted chromosomes shows structural problems. After bipolar attachment, the centromeric heterochromatin organized in the absence of Barren/CAP-H cannot withstand the forces exerted by the mitotic spindle and undergoes irreversible distortion. Taken together, our data suggest that the condensin I complex is required not only to promote sister chromatid resolution but also to maintain the structural integrity of centromeric heterochromatin during mitosis.     

The biological significance of variation in satellite DNA and heterochromatin in newts of the genus Triturus: an evolutionary perspective

The functional and evolutionary significance of highly repetitive, simple sequence (satellite) DNA is analysed by examining available information on the patterns of variation of heterochromatin and cloned satellites among newts (family Salamandridae), and particularly species of the European genus Triturus. This information is used to develop a model linking evolutionary changes in satellite DNAs and chromosome structure. In this model, satellites accumulate initially in large tandem blocks around centromeres of some or all of the chromosomes, mainly by repeated chromosomal exchanges in these regions. Centromeric blocks later become broken up and dispersed by small, random chromosome rearrangements in these regions. They are dispersed first to pericentric locations and then gradually more distally into the chromosome arms and telomeres. Dispersal of a particular satellite is accompanied by changes in sequence structure (for example, base substitutions, deletions, etc.) and a corresponding decrease in its detectability at either the molecular or cytological level. On the basis of this model, observed satellites in newt species may be classified as 'old', 'young', or of 'intermediate' phylogenetic age. The functions and effects of satellite DNA and heterochromatin at the cellular and organismal levels are also discussed. It is suggested that satellite DNA may have an impact on cell proliferation through the effect of late-replicating satellite-rich heterochromatin on the duration of S-phase of the cell cycle. It is argued that even small alterations in cell cycle time due to changes in heterochromatin amount may have magnified effects on organismal growth that may be of adaptive significance.     

Extraordinary conservation of entire chromosomes in insects over long evolutionary periods

Comparison of the genomes of different Drosophila species has shown that six different chromosomes, the so‐called ‘‘Muller elements,” constitute the building blocks for all Drosophila species. Here, we confirm previous results suggesting that this conservation of the Muller elements extends far beyond Drosophila, to at least tephritid fruit flies, thought to have diverged from drosophilids 60–70 mYr ago. Less than 10 percent of genes differ in chromosome location between the two insect groups. Within chromosomes, however, the order is highly scrambled, as expected from the comparison between Drosophila species. The data also support the notion that the sex chromosomes of tephritid flies originated from an ancestor of the dot chromosome 4 of Drosophila. Overall, therefore, no new chromosome has been created for perhaps a billion generations over the two evolutionary lines. This stability at the chromosome level, which appears to extend to all Diptera including mosquitoes, is in stark contrast to other groups such as mammals, birds, fish and plants, in which chromosome numbers and organization vary enormously among species that have diverged over much fewer generations.     

Chromosome and nucleotide sequence differentiation in genomes of polyploid Triticum species

Summary The nature of genome change during polyploid evolution was studied by analysing selected species within the tribe Triticeae. The levels of genome changes examined included structural alterations (translocations, inversions), heterochromatinization, and nucleotide sequence change in the rDNA regions. These analyses provided data for evaluating models of genome evolution in polyploids in the genus Triticum, postulated on the basis of chromosome pairing at metaphase I in interspecies hybrids.The significance of structural chromosome alterations with respect to reduced MI chromosome pairing in interspecific hybrids was assayed by determining the incidence of heterozygosity for translocations and paracentric inversions in the A and B genomes of T. timopheevii ssp. araraticum (referred to as T. araraticum) represented by two lines, 1760 and 2541, and T. aestivum cv. Chinese Spring. Line 1760 differed from Chinese Spring by translocations in chromosomes 1A, 3A, 4A, 6A, 7A, 3B, 4B, 7B and possibly 2B. Line 2541 differed from Chinese Spring by translocations in chromosomes 3A, 6A, 6B and possibly 2B. Line 1760 also differed from Chinese Spring by paracentric inversions in arms 1AL and 4AL whereas line 2541 differed by inversions in 1BL and 4AL (not all chromosomes arms were assayed). The incidence of structural changes in the A and B genomes did not coincide with the more extensive differentiation of the B genomes relative to the A genomes as reflected by chromosome pairing studies.To assay changing degrees of heterochromatinization among species of the genus Triticum, all the diploid and polyploid species were C-banded. No general agreement was observed between the amount of heterochromatin and the ability of the respective chromosomes to pair with chromosomes of the ancestral species. Marked changes in the amount of heterochromatin were found to have occurred during the evolution of some of the polyploids.The analysis of the rDNA region provided evidence for rapid fixation of new repeated sequences at two levels, namely, among the 130 bp repeated sequences of the spacer and at the level of the repeated arrays of the 9 kb rDNA units. These occurred both within a given rDNA region and between rDNA regions on nonhomologous chromosomes. The levels of change in the rDNA regions provided good precedent for expecting extensive nucleotide sequence changes associated with differentiation of Triticum genomes and these processes are argued to be the principal cause of genome differentiation as revealed by chromosome pairing studies.