José P. Abad

A dicentric chromosome of Drosophila melanogaster showing alternate centromere inactivation

Abstract.Dicentric chromosomes are rarely found, because they interfere with normal cell division causing chromosome instability. By in situ hybridization of region-specific heterochromatic yeast artificial chromosomes we have found that the artificially generated C(1)A chromosome of Drosophila melanogaster has two potential centromeres: one carries all the sequences of the centromere of the Y chromosome and the other carries only a part of the Y centromeric region that is rich in telomere-related sequences. Immunostaining with anti-Bub1 (a kinetochore-specific marker) shows that, in spite of the differences in sequence, both centromeres can be active although as a rule only one at a time. In a small fraction of the chromosomes centromere inactivation is incomplete, giving rise to true dicentric chromosomes. The centromere inactivation is clonally inherited, providing a new example of epigenetic chromosome imprinting and the possibility of genetically dissecting this process. The involvement of telomere-related sequences in centromere function is discussed.

Centromeric dodeca-satellite DNA sequences form fold-back structures.

The evolutionarily conserved centromeric dodeca-satellite DNA has an asymmetric distribution of guanine and cytosine residues resulting in one strand being relatively G-rich. This dodeca-satellite G-strand contains a GGGA-tract that is similar to the homopurine tracts found in most telomeric DNA sequences. Here, we show that the dodeca-satellite G-strand forms intramolecular hairpin structures that are stabilized by the formation of non-Watson-Crick G.A pairs as well as regular Watson-Crick G.C pairs. Special stacking interactions are also likely to contribute significantly to the stability of this structure. This hairpin conformation melts at relatively high temperature, around 75 degrees C, and is detected under many different ionic and pH conditions. As judged by electron microscopy visualization, these structures can be formed in a B-DNA environment. Under the same experimental conditions, neither the C-strand nor the double-stranded dodeca-satellite DNA were found to form any unusual DNA structure. A protein activity has been detected that preferentially binds to the single-stranded dodeca-satellite C-strand. The biological relevance of these results is discussed in view of the similarities to telomeric DNA.

Organization of DNA sequences near the centromere of the Drosophila melanogaster Y chromosome.

Abstract. The structural analysis of a yeast artificial chromosome clone from Drosophila melanogaster enriched in dodecasatellite sequences has led us to find a new retrotransposon that we have called Circe. Moreover, this retrotransposon has allowed the isolation of a contig encompassing ∼200 kb near the centromere of the Y chromosome, providing an entry point into a region from which very little sequence information has been obtained to date. The molecular characterization of the contig has shown the presence of HeT-A telomeric retrotransposons close to the centromere of the Y chromosome, suggesting a telocentric origin for this submetacentric chromosome.

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.

The 3′ non-coding region of the Drosophila melanogaster HeT-A telomeric retrotransposon contains sequences with propensity to form G-quadruplex DNA

HeT‐A elements are non‐long terminal repeat retrotransposons added onto the Drosophila chromosome ends. We have investigated the formation in vitro of higher order structures by oligonucleotides derived from the 3′ non‐coding region of HeT‐A elements and found that they are capable of forming G‐quadruplex DNA. These results suggest that the 3′ repeat region of HeT‐A may structurally behave as the telomeric repeats common to a majority of eukaryotes. The presence of structural motifs shared by telomeres and centromeres and the implications of these findings for chromosome evolution are discussed.

Searching for a common centromeric structural motif: Drosophila centromeric satellite DNAs show propensity to form telomeric-like unusual DNA structures.

The molecular basis of centromere formation in a particular chromosomal region is not yet understood. In higher eukaryotes, no specific DNA sequence is required for the assembly of the kinetochore, but similar centromeric chromatins are formed on different centromere DNA sequences. Although epigenesis has been proposed as the main mechanism for centromere specification, DNA recognition must also play a role. Through the analysis of Drosophilacentromeric DNA sequences, we found that dodeca satellite and 18HT satellite are able to form unusual DNA structures similar to those formed by telomeric sequences. These findings suggest the existence of a common centromeric structural DNA motif which we feel merits further investigation.

Genomic organization of the metal-mobilizing bacterium Thiobacillus cuprinus.

The genomic organization of Thiobacillus cuprinus, a facultative chemolithotrophic bacterium that preferentially solubilizes copper from complex ores, has been studied by Pulsed Field Gel Electrophoresis (PFGE). It has been determined that T. cuprinus has a circular chromosome of about 3.8 Mb in size as concluded by analysis of gamma-irradiated total DNA and restriction analysis. Macrorestriction patterns for several restriction enzymes have been generated. Restriction enzymes AseI, DraI, SpeI, SwaI and XbaI give a number of fragments that can be adequately resolved by PFGE and then be used for electrophoretic karyotyping and for the construction of physical maps of the chromosome. Such a map has been constructed for the endonucleases SpeI and SwaI. The localization of several heterologous and homologous genes on the physical map, including those for rRNA, has paved the way for the construction of a genetic map.