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J P Abad

Publications and source records attributed to J P Abad.

At least 19 recordsLinked to original sources
PubMed2001

Preparation of DNA suitable for PCR amplification from fresh or fixed single dinoflagellate cells.

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).

Animals↗
PubMed2000

A dicentric chromosome of Drosophila melanogaster showing alternate centromere inactivation.

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.

Animals↗
PubMed2000

Pericentromeric regions containing 1.688 satellite DNA sequences show anti-kinetochore antibody staining in prometaphase chromosomes of Drosophila melanogaster.

A striking characteristic of the centromeric heterochromatin of Drosophila melanogaster is that each chromosome carries different satellite DNA sequences. Here we show that while the major component of the 1.688 satellite DNA family expands across the centromere of the X chromosome the rest of the minor variants are located at pericentromeric positions in the large autosomes. Immunostaining of prometaphase chromosomes with the kinetocore-specific anti-BUB1 antibody reveals the transient presence of this centromeric protein in all the regions containing the 1.688 satellite.

Animals↗
PubMed2000

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 Drosophila centromeric 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.

Animals↗
PubMed1999

Centromeres from telomeres? The centromeric region of the Y chromosome of Drosophila melanogaster contains a tandem array of telomeric HeT-A- and TART-related sequences.

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.

Animals↗
PubMed1999

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.

Animals↗
PubMed1999

HeT-A telomere-specific retrotransposons in the centric heterochromatin of Drosophila melanogaster chromosome 3.

We have isolated two yeast artificial chromosome (YAC) clones from Drosophila melanogaster that contain a small amount of dodeca satellite (a satellite DNA located in the centromeric region of chromosome 3) and sequences homologous to the telomeric retro-transposon HeT-A. Using these YACs as probes for fluorescence in situ hybridization to mitotic chromosomes, we have localized these HeT-A elements to the centric heterochromatin of chromosome 3, at region h55. The possible origin of these telomeric elements in a centromeric position is discussed.

Animals↗
PubMed1999

The analysis of Circe, an LTR retrotransposon of Drosophila melanogaster, suggests that an insertion of non-LTR retrotransposons into LTR elements can create chimeric retroelements.

Circe is a transposable element recently identified in Drosophila melanogaster which appears to be mostly associated with the constitutive heterochromatin. This element shows the structural features of a long terminal repeat (LTR)-containing retrotransposon: It is flanked by 240-bp-long terminal repeats, and its two open reading frames encode putative proteins resembling the gag and pol polyproteins of retroviruses. However, Circe displays striking similarities of both LOA and Ulysses, a non-LTR element and an LTR element, respectively. The result of its phylogenetic and structural analysis has allowed us to propose a new mechanism for non-LTR retrotransposon evolution.

Amino Acid Sequence↗
PubMed1998

Genomic organization analysis of acidophilic chemolithotrophic bacteria using pulsed field gel electrophoretic techniques.

The genomic organization of acidophilic chemolithotrophic bacteria belonging to the genus Thiobacillus, Thiomonas and Leptospirillum was studied using pulsed field gel electrophoresis techniques (PFGE). The electrophoretic analysis of intact DNA prepared from different strains showed that all have a circular chromosome, with sizes ranging from 1.9 Mb for Leptospirillum ferrooxidans ATCC 49879, the smallest genome for an acidophilic strict chemolithoautotrophic microorganism, to 3.8 Mb for Thiomonas cuprina DSM 5495, the largest in this study. The number of extrachromosomal elements present varied from none, as observed in several isolates of Leptospirillum ferrooxidan, to five in Thiobacillus thiooxidans ATCC 8085. The mixotroph Thiomonas cuprina DSM 5495 was found to have a linear 50 kb megaplasmid which was inducible when the bacteria was grown in chemolithotrophic conditions. Low-frequency restriction fragment analysis (LFRFA) of different acidophilic chemolithotrophs and related species was carried out by PFGE to determine macrorestriction patterns for rare cutters (SpeI, XbaI, SwaI, PmeI), which were then used for taxonomic identification (karyotyping), genome size determination, and generation of physical and genetic maps.

Chromosome Mapping↗
PubMed1997

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.

Chromosomes↗
PubMed1997

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

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 approximately 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.

Animals↗
PubMed1995

High-ionic strength interference of ribosomal inhibition produced by aminoglycoside antibiotics.

A protein synthesis cell-free system capable of performing with similar efficiencies in different ionic conditions has been developed for the halotolerant marine bacterium Vibrio costicola. The system has been used to test the effect of ionic strength on the interference produced by thirty translation inhibitors with different structural, functional, and domain specificities. In general, at high ionic strengths, the inhibition of protein synthesis produced by polycationic antibiotics like the aminoglycosides is much less pronounced than the inhibition obtained at low ionic strengths, while non-aminoglycosidic antibiotics show similar inhibitory activities at both high and low ionic conditions. These results strongly suggest that competition between polycationic antibiotics and cations at high concentrations in the media is responsible for the lack of inhibition by aminoglycoside antibiotics at high ionic strengths, rather than a lack of binding sites.

Aminoglycosides↗
PubMed1995

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.

Animals↗
PubMed1994

Location of the streptomycin ribosomal binding site explains its pleiotropic effects on protein biosynthesis.

Photoaffinity-labeling experiments using three nitroguaiacol ether streptomycin derivatives with spacers of different lengths between the antibiotic and the photoreactive moiety (8, 12 and 17 A) allow us to: (1) unambiguously locate the boundaries of the antibiotic binding site; and (2) test the topographical consistency of the photolabeling results. The streptomycin binding site is located in the interface between the ribosomal subunits, close to proteins S5 in the 30 S and to L11 in the 50 S ribosomal subunits. This location explains most of the antibiotic's pleiotropic effects on protein biosynthesis, especially those related to the tRNA selection mechanism, and it also correlates with the location of the ribosomal components involved in the different streptomycin phenotypes.

Affinity Labels↗
PubMed1994

Halobacterial megaplasmids are negatively supercoiled.

Several covalently closed circular halobacterial megaplasmids (up to more than 500 kb) from different strains of Haloferax mediterranei, have been resolved by orthogonal-field alternating gel electrophoresis (OFAGE). These molecules seem to be negatively supercoiled in vivo, as deduced from the effect of intercalating agents affecting their topology and, therefore, their electrophoretic mobility. It has also been demonstrated that the topoisomerase II inhibitor novobiocin affects the native topological state of halobacterial megaplasmids impeding their migration in OFAGE under standard conditions for resolution of large supercoiled molecules.

DNA, Superhelical↗
PubMed1993

The Drosophila melanogaster dodecasatellite sequence is closely linked to the centromere and can form connections between sister chromatids during mitosis.

We have used fluorescence in situ hybridisation to wild-type and rearranged mitotic chromosomes to map the Drosophila melanogaster dodecasatellite sequence. It is located at a unique site, within the pericentric heterochromatin of the right arm of the third chromosome, closely linked to the primary constriction. In polytene chromosomes, dodecasatellite is found as one or a few dots in the central region of the chromocentre. In untreated diploid cells, dodecasatellite sequences are found as one or two dots throughout the cell cycle. This distribution can be altered in a cell cycle-dependent manner in two ways. Firstly, in interphase cells, hypotonic shock promotes the decondensation of the genomic region containing this satellite, resulting in a string-like structure. Secondly, some of the precociously separated sister chromatids produced by colchicine treatment show dodecasatellite within the intervening space connecting the main dodecasatellite signals of each chromatid. The distribution of dodecasatellite seems to be rather constant between individuals of the same species, as indicated by the lack of any detectable variations in its pattern amongst individuals from six geographically distant strains of D. melanogaster. On the other hand, the distribution of dodecasatellite shows a remarkable degree of variation amongst closely related species of the melanogaster subgroup ranging from a non-detectable signal in Drosophila yakuba and Drosophila teissieri, to staining in the X, second and third chromososomes of Drosophila mauritiana.

Animals↗
PubMed1992

Genomic organization of the halophilic archaeon Haloferax mediterranei: physical map of the chromosome.

Pulsed field gel electrophoresis (PFG) has been used to study the genomic organization of the halophilic archaeon Haloferax mediterranei. Analysis of the different genomic elements as well as the restriction patterns obtained with several endonucleases revealed that this microorganism has a circular chromosome of 2.9 Mb and, at least, three extrachromosomal elements of 490, 320 and 130 kb respectively. The complete physical map of the chromosome for the endonucleases PacI and BamHI has been constructed, and several BcII, BgIII and DraI restriction fragments have been aligned on these maps. The localization of heterologous and homologous genes on the physical map, including those for rRNA, lay the ground work for the construction of a genetic map.

Chromosomes, Bacterial↗