Beatriz de Pablos

Mapping and identification of essential gene functions on the X chromosome of Drosophila

The Drosophila melanogaster genome consists of four chromosomes that contain 165 Mb of DNA, 120 Mb of which are euchromatic. The two Drosophila Genome Projects, in collaboration with Celera Genomics Systems, have sequenced the genome, complementing the previously established physical and genetic maps. In addition, the Berkeley Drosophila Genome Project has undertaken large‐scale functional analysis based on mutagenesis by transposable P element insertions into autosomes. Here, we present a large‐scale P element insertion screen for vital gene functions and a BAC tiling map for the X chromosome. A collection of 501 X‐chromosomal P element insertion lines was used to map essential genes cytogenetically and to establish short sequence tags (STSs) linking the insertion sites to the genome. The distribution of the P element integration sites, the identified genes and transcription units as well as the expression patterns of the P‐element‐tagged enhancers is described and discussed.

The Release 6 reference sequence of the Drosophila melanogaster genome

Drosophila melanogaster plays an important role in molecular, genetic, and genomic studies of heredity, development, metabolism, behavior, and human disease. The initial reference genome sequence reported more than a decade ago had a profound impact on progress in Drosophila research, and improving the accuracy and completeness of this sequence continues to be important to further progress. We previously described improvement of the 117-Mb sequence in the euchromatic portion of the genome and 21 Mb in the heterochromatic portion, using a whole-genome shotgun assembly, BAC physical mapping, and clone-based finishing. Here, we report an improved reference sequence of the single-copy and middle-repetitive regions of the genome, produced using cytogenetic mapping to mitotic and polytene chromosomes, clone-based finishing and BAC fingerprint verification, ordering of scaffolds by alignment to cDNA sequences, incorporation of other map and sequence data, and validation by whole-genome optical restriction mapping. These data substantially improve the accuracy and completeness of the reference sequence and the order and orientation of sequence scaffolds into chromosome arm assemblies. Representation of the Y chromosome and other heterochromatic regions is particularly improved. The new 143.9-Mb reference sequence, designated Release 6, effectively exhausts clone-based technologies for mapping and sequencing. Highly repeat-rich regions, including large satellite blocks and functional elements such as the ribosomal RNA genes and the centromeres, are largely inaccessible to current sequencing and assembly methods and remain poorly represented. Further significant improvements will require sequencing technologies that do not depend on molecular cloning and that produce very long reads.

Transcription of the 1.688 Satellite DNA Family Is under the Control of RNA Interference Machinery in Drosophila Melanogaster Ovaries

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-E1 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.

Telomere maintenance in Drosophila: rapid transposon evolution at chromosome ends.

The maintenance of terminal sequences is an important role of the telomere, since it prevents the loss of internal regions that encode essential genes. In most eukaryotes, this is accomplished by the telomerase. However, telomere length can also be maintained by other mechanisms, such as homologous recombination and transposition of telomeric retrotransposons to the chromosome ends. A remarkable situation is the case of Drosophila, where telomerase was lost, and thus telomeres managed to be maintained by occasional retrotransposition of telomeric elements to the receding ends. In the recent analysis of 12 Drosophila genomes, ¬¬the multiplicity of autonomous and non-autonomous telomere-specific retrotransposons has revealed extensive and rapid evolution of telomeric DNA. The phylogenetic relationship among these telomeric retrotransposons is congruent with the species phylogeny, suggesting that they have been vertically transmitted from a common ancestor. In this review, we also suggest that the formation of a non-canonical DNA structure at Drosophila telomeres could be the way to protect the ends.

Novel sequencing strategy for repetitive DNA in a Drosophila BAC clone reveals that the centromeric region of the Y chromosome evolved from a telomere

The centromeric and telomeric heterochromatin of eukaryotic chromosomes is mainly composed of middle-repetitive elements, such as transposable elements and tandemly repeated DNA sequences. Because of this repetitive nature, Whole Genome Shotgun Projects have failed in sequencing these regions. We describe a novel kind of transposon-based approach for sequencing highly repetitive DNA sequences in BAC clones. The key to this strategy relies on physical mapping the precise position of the transposon insertion, which enables the correct assembly of the repeated DNA. We have applied this strategy to a clone from the centromeric region of the Y chromosome of Drosophila melanogaster. The analysis of the complete sequence of this clone has allowed us to prove that this centromeric region evolved from a telomere, possibly after a pericentric inversion of an ancestral telocentric chromosome. Our results confirm that the use of transposon-mediated sequencing, including positional mapping information, improves current finishing strategies. The strategy we describe could be a universal approach to resolving the heterochromatic regions of eukaryotic genomes.

Genomic and cytological analysis of the Y chromosome of Drosophila melanogaster: telomere-derived sequences at internal regions

The genomic analysis of heterochromatin is essential for studying chromosome behavior as well as for understanding chromosome evolution. The Y chromosome of Drosophila melanogaster is entirely heterochromatic and the under-representation of this chromosome in genomic libraries together with the difficulty of assembling its sequence has made its study very difficult. Here, we present the construction of bacterial artificial chromosome (BAC) contigs from regions h14, h16 and the centromeric region h18. The analysis of these contigs shows that telomere-derived sequences are present at internal regions. In addition, immunostaining of prometaphase chromosomes with an antibody to the kinetochore-specific protein BubR1 has revealed the presence of this protein in some Y chromosome regions rich in telomere-related sequences. Collectively, our data provide further evidence for the hypothesis that the Drosophila Y chromosomes might have evolved from supernumerary chromosomes.

A Large Palindrome With Interchromosomal Gene Duplications in the Pericentromeric Region of the D. melanogaster Y Chromosome

The non-recombining Y chromosome is expected to degenerate over evolutionary time, however, gene gain is a common feature of Y chromosomes of mammals and Drosophila. Here, we report that a large palindrome containing interchromosomal segmental duplications is located in the vicinity of the first amplicon detected in the Y chromosome of D. melanogaster. The recent appearance of such amplicons suggests that duplications to the Y chromosome, followed by the amplification of the segmental duplications, are a mechanism for the continuing evolution of Drosophila Y chromosomes.