Comparative analysis of multiple genomes in a phylogenetic framework dramatically improves the precision and sensitivity of evolutionary inference, producing more robust results than single-genome analyses can provide. The genomes of 12 Drosophila species, ten of which are presented here for the first time (sechellia, simulans, yakuba, erecta, ananassae, persimilis, willistoni, mojavensis, virilis and grimshawi), illustrate how rates and patterns of sequence divergence across taxa can illuminate evolutionary processes on a genomic scale. These genome sequences augment the formidable genetic tools that have made Drosophila melanogaster a pre-eminent model for animal genetics, and will further catalyse fundamental research on mechanisms of development, cell biology, genetics, disease, neurobiology, behaviour, physiology and evolution. Despite remarkable similarities among these Drosophila species, we identified many putatively non-neutral changes in protein-coding genes, non-coding RNA genes, and cis-regulatory regions. These may prove to underlie differences in the ecology and behaviour of these diverse species.

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To identify sequences from the centromeric region, we have constructed a Drosophila melanogaster yeast artificial chromosome (YAC) library and screened it with purified DNA from the minichromosome Dp(l;f)1187 derived from the X chromosome. We describe the structure ofone clone isolated in this way. This YAC is structurally unstable and contains tandemly repeated G+C-rich li-mer and 12-mer units, which we call dodeca satellite. Most of this satellite is located near the centromere of an autosome. Cross-hybridizing sequences are found in the genomes of organisns as distant as Arabidopsis thaliana and Homo sapiens.A total of 11 simple repeated sequences have been cloned from gradient-purified satellite DNA ofD. melanogaster and the nucleotide sequence ofeach repeat conforms to a formula (RRN)m(RN),,, where R is A or G and N is any nucleotide (8).In this study, we have developed an approach to the cloning of centromeric heterochromatin sequences from D. melanogaster. Thus, we have discovered a type of tandemly repeated DNA sequence that is located in the centromeric region of some D. melanogaster chromosomes and that cross-hybridizes with DNA from other species including Arabidopsis and humans.** The genomes of higher eukaryotes contain large amounts of simple and complex tandemly repeated DNA sequences, classically termed satellite DNA (for review, see ref.

The centromere is the DNA region of the eukaryotic chromosome that determines kinetochore formation and sister chromatid cohesion. Centromeres interact with spindle microtubules to ensure the segregation of chromatids during mitosis and of homologous chromosomes in meiosis. The origin of centromeres, therefore, is inseparable from the evolution of cytoskeletal components that distribute chromosomes to offspring cells. Although the origin of the nucleus has been debated, no explanation for the evolutionary appearance of centromeres is available. We propose an evolutionary scenario: The centromeres originated from telomeres. The breakage of the ancestral circular genophore activated the transposition of retroelements at DNA ends that allowed the formation of telomeres by a recombination-dependent replication mechanism. Afterward, the modification of the tubulin-based cytoskeleton that allowed specific subtelomeric repeats to be recognized as new cargo gave rise to the first centromere. This switch from actin-based genophore partition to a tubulin-based mechanism generated a transition period during which both types of cytoskeleton contributed to fidelity of chromosome segregation. During the transition, pseudodicentric chromosomes increased the tendency toward chromosomal breakage and instability. This instability generated multiple telocentric chromosomes that eventually evolved into metacentric or holocentric chromosomes. C entromeres are typically composed of rapidly evolving satellite DNA sequences; therefore, centromeric DNA is not broadly conserved throughout evolution. However, in agreement with the conserved centromeric function, many centromere/kinetochore proteins are highly conserved. This apparent paradox can be explained by the coevolution of kinetochore proteins and centromeric DNA sequences as is apparent with the centromere-specific histone H3 variant CENP-A in Drosophila and Arabidopsis (1, 2). The periodic homogenization/ amplification undergone by any tandemly repeated DNA sequence via unequal crossing over (3, 4) provides a mechanism for new sequences to be amplified that are specific for novel kinetochore protein variants. Such a progressive substitution of tandemly repeated DNA sequences would explain the lack of discernible homology between centromeric sequences in different organisms. Nevertheless, it is also possible that a detailed study of centromeric sequences could lead to the identification of a common sequence-independent structural recognition determinant within centromeric DNA. Although such a conserved structural motif might direct the formation of centromeric chromatin on its own, the episodic occurrence of centromere activity associated with noncentromeric sequences, neocentromeres (5, 6), and the frequent inactivation/activation of centromeric structures (7-10) indicate that centromere specification involves an epigenetic mechanism that recognizes some characteristic of centromeric DNA (11).The comparative analysis of the centromeres from Drosophila melanogaster, vertebrates, a...

Cytological and cytogenetic studies have previously defined the region needed for centromeric function in the Y chromosome of Drosophila melanogaster. We have identified a YAC clone that originated from this region. Molecular analysis of the YAC and genomic DNAs has allowed the description of a satellite DNA made of telomeric HeT-A- and TART-derived sequences and the construction of a long-range physical map of the heterochromatic region h18. Sequences within the YAC clone are conserved in the centromeric region of the sibling species Drosophila simulans. That telomere-derived DNA now forms part of the centromeric region of the Y chromosome could indicate a telomeric origin of this centromere. The existence of common determinants for the function of both centromeres and telomeres is discussed.

Here we show that RNA interference (RNAi) machinery operates in Drosophila melanogaster 1.688 satellite transcription. Mutation in the spn-E gene, known to be involved in RNAi in the oocytes, causes an increase of satellite transcript abundance. Transcripts of both strands of 1.688 satellite repeats in germinal tissues were detected. The strength of the effects of the spn-E mutation differs for 1.688 satellite DNA subfamilies and is more pronounced for autosomal pericentromeric satellites compared to the X-linked centromeric ones. The spn-E 1 mutation causes an increase of the H3-AcK9 mark and TAF1 (a component of the polymerase II transcriptional complex) occupancy in the chromatin of autosomal pericentromeric repeats. Thus, we revealed that RNAi operates in ovaries to maintain the silenced state of centromeric and pericentromeric 1.688 repeats.

A method is described to prepare total DNA from single cells of dinoflagellates, which can be used for PCR amplification. As model organisms, we used a stock strain of Alexandrium catenella and cells of Dinophysis acuminata harvested from the Atlantic Ocean. Fresh grown cells or cells maintained in different preservatives were tested as sources for DNA preparation. The method used to prepare DNA combines physicochemical and enzymatic procedures on cells embedded in agarose plugs or beads. The agarose pieces containing the DNA were used to perform PCR amplification of a fragment of DNA containing a 5.8S rRNA gene and the flanking internal transcribed spacers (ITS1 and ITS2).

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