PubMed Health⌕ Search

Biomedical subjects

D de Bruin

Publications and source records attributed to D de Bruin.

At least 19 recordsLinked to original sources

Kinetic physical etching for versatile novel design of well ordered self-affine nanogrooves.

Ion bombardment at extreme grazing incidence leads to the formation of remarkably well ordered, one to two atomic layer deep, parallel grooves on a Cu(001) surface. These self-organized grooves are oriented parallel to the ion's plane of incidence and their period can be controlled between approximately 4 and 15 nm. We have identified two distinct temperature regimes: Between about 200 and 300 K the substrate temperature controls the period, while below 200 to about 150 K the ion energy does. The groove separation distribution shows distinct self-affine character. The physical origin of the novel nanostructures is discussed.

Journal Article↗

Telomere looping permits gene activation by a downstream UAS in yeast.

In yeast (Saccharomyces cerevisiae), transcriptional activators, such as Gal4 and Gal4-VP16, work ordinarily from sites located in the upstream activating sequence (UAS) positioned about 250 base pairs upstream of the transcription start site. In contrast to their behaviour in mammalian cells, however, such activators fail to work when positioned at distances greater than approximately 600-700 base pairs upstream, or anywhere downstream of the gene. Here we show that, in yeast, a gene bearing an enhancer positioned 1-2 kilobases downstream of the gene is activated if the reporter is linked to a telomere, but not if it is positioned at an internal chromosomal locus. These observations are explained by the finding that yeast telomeres form back-folding, or looped, structures. Because yeast telomeric regions resemble the heterochromatin found in higher eukaryotes, these findings might also explain why transcription of some higher eukaryotic genes depends on their location in heterochromatin.

Chromatin↗

Cognitive performance and serotonergic function in users of ecstasy.

RATIONALE: (+/-) 3,4-Methylenedioxymethamphetamine (MDMA or "ecstasy") has been shown to cause long term damage to serotonergic cerebral neurons in animals. The neurotoxic effects in humans are less clear and little is known about the functional consequences, although some studies suggest memory impairment. Given the widespread use of MDMA, our lack of knowledge raises concerns. OBJECTIVE: We investigated, in humans, the relation between past use of ecstasy and cognitive performance as well as serotonergic function. METHODS: Two groups of 21 males with moderate and heavy recreational use of MDMA, respectively, and a control group of 20 males without use of MDMA were compared. All were from the same subculture. Reaction time, direct recall, and recognition were assessed. Serotonergic function was measured by the neuro-endocrine response to a placebo-controlled, crossover challenge with dexfenfluramine. RESULTS: Ecstasy users showed a broad pattern of statistically significant, but clinically small, impairment of memory and prolonged reaction times. Heavy users were affected stronger than moderate users. Release of cortisol but not of prolactin after dexfenfluramine administration was significantly reduced in both groups of ecstasy users compared with the controls. Analyses of covariance showed that likely confounding variables including recent exposure to ecstasy, psychosocial profiles and use of other drugs did not explain the differences found between the groups. CONCLUSIONS: These results provide further evidence that use of ecstasy may be associated with impairment of memory and of serotonergic function. These findings are compatible with neurotoxicity of ecstasy as shown in animals.

Adolescent↗

Telomere folding is required for the stable maintenance of telomere position effects in yeast.

Yeast telomeres reversibly repress the transcription of adjacent genes, a phenomenon called telomere position effect (TPE). TPE is thought to result from Rap1 and Sir protein-mediated spreading of heterochromatin-like structures from the telomeric DNA inwards. Because Rap1p is associated with subtelomeric chromatin as well as with telomeric DNA, yeast telomeres are proposed to form fold-back or looped structures. TPE can be eliminated in trans by deleting SIR genes or in cis by transcribing through the C(1-3)A/TG(1-3) tract of a telomere. We show that the promoter of a telomere-linked URA3 gene was inaccessible to restriction enzymes and that accessibility increased both in a sir3 strain and upon telomere transcription. We also show that subtelomeric chromatin was hypoacetylated at histone H3 and at each of the four acetylatable lysines in histone H4 and that histone acetylation increased both in a sir3 strain and when the telomere was transcribed. When transcription through the telomeric tract occurred in G(1)-arrested cells, TPE was lost, demonstrating that activation of a silenced telomeric gene can occur in the absence of DNA replication. The loss of TPE that accompanied telomere transcription resulted in the rapid and efficient loss of subtelomeric Rap1p. We propose that telomere transcription disrupts core heterochromatin by eliminating Rap1p-mediated telomere looping. This interpretation suggests that telomere looping is critical for maintaining TPE.

Acetylation↗

The yeast Cac1 protein is required for the stable inheritance of transcriptionally repressed chromatin at telomeres.

Cac1p is a subunit of yeast chromatin assembly factor I (yCAF-I) that is thought to assemble nucleosomes containing diacetylated histones onto newly replicated DNA [Kaufman, P. D., Kobayashi, R. & Stillman, B. (1997) Genes Dev. 11, 345-357]. Although cac1 delta cells could establish and maintain transcriptional repression at telomeres, they displayed a reduced heritability of the repressed state. Single-cell analysis revealed that individual cac1 delta cells switch from transcriptionally "off" to transcriptionally "on" more often per cell cycle than wild-type cells. In addition, cac1 delta cells were defective for transcriptional silencing near internal tracts of C(1-3)A sequence, but they showed no defect in silencing at the silent mating type loci when analyzed by a reverse transcription-PCR assay. Despite the loss of transcriptional silencing at telomeres and internal C(1-3)A tracts, subtelomeric DNA was organized into nucleosomes that had all of the features characteristic of silent chromatin, such as hypoacetylation of histone H4 and protection from methylation by the Escherichia coli dam methylase. Thus, these features of silent chromatin are not sufficient for stable maintenance of a silent chromatin state. We propose that the inheritance of the transcriptionally repressed state requires the specific pattern of histone acetylation conferred by yCAF-I-mediated nucleosome assembly.

Chromatin↗

A YAC contig map of Plasmodium falciparum chromosome 4: characterization of a DNA amplification between two recently separated isolates.

We have generated a physical map of Plasmodium falciparum chromosome 4 using yeast artificial chromosomes (YACs). The map is defined by a YAC contig spanning approximately 1.05 Mb, which has been restriction mapped to a resolution of 30 kb and is punctuated by 22 sequence-tagged sites. The physical information obtained has enabled us to compare and contrast the structure of chromosome 4 in detail between FCR3 and B8, two recently separated isolates of P. falciparum, leading to characterization of a novel chromosome polymorphism occurring in a subtelomeric region. Comparison of chromosomes 4 from 10 different isolates has shown that chromosome size polymorphisms are restricted to both subtelomeric regions. These analyses provide a high-resolution physical map that will be important to complement genetic analysis of this human pathogen.

Animals↗

Transcriptional and nucleosomal characterization of a subtelomeric gene cluster flanking a site of chromosomal rearrangements in Plasmodium falciparum.

We have recently demonstrated that Plasmodium falciparum chromosomes are compartmentalized into different domains: conserved and polymorphic domains; transcriptionally active and silent domains. Here we have analyzed the transition between these domains at the structural and nucleosomal level. This study was conducted with the end of chromosome 2 that is associated with cytoadherence. At this end of the chromosome, the first set of erythrocytic genes has been mapped 85 kb from the telomere. These genes are monocistronically transcribed as revealed by nuclear run-on analysis. Two of these genes, PfEMP3 and KAHRP, are deleted in cytoadherent negative mutants, whereas the third gene, GLARP, is expressed in all the strains investigated. The data indicate that the polymorphic domain at this end of chromosome 2 extends into the transcribed region. Analysis of the chromatin structure revealed that both the transcribed domain and the subtelomeric region are organized as nucleosomes with a periodicity of 155 +/- 5 bp.

Animals↗

Chromatin structure determines the sites of chromosome breakages in Plasmodium falciparum.

Spontaneous chromosome breakages are frequently observed in the human malaria parasite Plasmodium falciparum and are responsible for the generation of novel phenotypes, which may contribute to the pathogenicity and virulence of this protozoan parasite. The identification of a hot spot of chromosome breakage within the coding region of the KAHRP gene revealed that these events do not occur randomly but follow a regular pattern with a periodicity of 155 bp. This phasing corresponds to the average repeat unit of P. falciparum nucleosomes. Furthermore, breakage events preferentially occur within the linker regions of nucleosomes, as demonstrated by mapping endonuclease hypersensitive sites of chromatin. These data suggest that, in P. falciparum, the chromatin structure is involved in the molecular process of chromosome breakage, a mechanism that may be common in other eukaryotes.

Animals↗

The polymorphic subtelomeric regions of Plasmodium falciparum chromosomes contain arrays of repetitive sequence elements.

The human malaria parasite Plasmodium falciparum exhibits a high degree of chromosomal polymorphism, which may contribute to its ability to evade host defenses. The analysis of parasite chromosomes has revealed that these polymorphisms are confined to the subtelomeric regions, which are transcriptionally silent and contain repetitive sequence elements. Several subtelomeric repetitive elements have been isolated and mapped by using P. falciparum yeast artificial chromosome (YAC) clones. Structural analysis of parasite telomeric and subtelomeric YAC clones demonstrated that these repetitive elements are conserved between P. falciparum chromosome ends. We suggest that these subtelomeric elements promote chromosome pairing in P. falciparum and facilitate meiotic recombination and gene conversion between telomere-proximal genes.

Animals↗

Organization of chromosomes in Plasmodium falciparum: a model for generating karyotypic diversity.

Chromosomes of the human malaria parasite, Plasmodium falciparum, are compartmentalized into conserved and polymorphic domains. The conserved domains are transcribed and localized within the central region o f a chromosome, whereas the polymorphic domains are transcriptionally silent and situated at chromosome ends. Unlike the central chromosomal domains, chromosome ends contain repetitive sequence elements. In this review, Michael Lonzer, Derik de Bruin, Samuel Wertheimer and Jeffrey Ravetch propose that the chromosome ends play a functional role in generating genetic diversity by promoting meiotic and mitotic recombination and chromosomal rearrangement events.

Journal Article↗

Transcriptional differences in polymorphic and conserved domains of a complete cloned P. falciparum chromosome.

Classical genetic studies on the human malaria parasite Plasmodium falciparum have been hampered by a complex life cycle which alternates between vertebrate and invertebrate hosts. Consequently, only a few genetic crosses have been performed so far. In addition, molecular genetics has provided only limited access to the genes of this pathogen, a consequence of an unusually high A + T content. To overcome these limitations we have constructed an ordered telomere-to-telomere contig map of P. falciparum chromosome 2 by isolating overlapping yeast artificial chromosome clones. This approach was used to examine the strain-dependent polymorphisms commonly observed for P. falciparum chromosomes. Our analysis reveals that polymorphisms of chromosome 2 are restricted to regions at either end, representing 20% of the chromosome. Transcription mapping of the entire chromosome suggests a compartmentalization of chromosome 2 into a transcribed central domain and silent polymorphic ends.

Animals↗

Transcriptional diversity at the duplicated human CD8 beta loci.

In order to understand the structural organization of the human CD8 beta locus, genomic clones containing CD8 beta sequences were isolated and analyzed. Physical linkage of these clones with the CD8 alpha locus using pulsed-field electrophoresis revealed a duplication of the CD8 beta locus. CD8B-1 lies 35 kb upstream from the CD8 alpha locus and contains eight exons, including four alternatively spliced cytoplasmic exons. The CD8B-2 gene contains six exons and is at present unlinked to CD8B-1. Analysis of sequences upstream to the leader exon of the CD8B-1 and CD8B-2 genes revealed a GC-rich promoter which lacks canonical "CCAAT" and "TATA" motifs, but which has sites for multiple transcriptional activators and three additional elements which are conserved in the murine CD8 beta promoter. Seven unique CD8 beta cDNA isoforms were isolated and characterized, which derive from alternative splicing of the transmembrane and/or cytoplasmic exons. Three cDNA are membrane spanning, while the remaining four isoforms lack a transmembrane region and are potentially secreted. These transcripts are differentially expressed in the thymus and in the periphery. Transfection experiments in murine fibroblasts confirmed that the membrane CD8 beta isoforms could be expressed as heterodimers with the CD8 alpha chain. The regulated expression of multiple CD8 beta cytoplasmic isoforms and their potential role in T lymphocyte signal transduction is discussed.

Alternative Splicing↗

A sequence element associated with the Plasmodium falciparum KAHRP gene is the site of developmentally regulated protein-DNA interactions.

The Plasmodium falciparum gene encoding the knob associated histidine-rich protein (KAHRP) is shown to be transcriptionally regulated during its expression in the intraerythrocytic cycle as demonstrated by stage specific nuclear run-on analysis. The genomic organization of the KAHRP gene was determined and the structural basis for the stage specific transcription investigated. A sequence motif with two-fold symmetry was found 160 bp upstream of the RNA initiation site. This sequence element interacts with parasite derived nuclear extracts in a stage specific manner that correlates with the transcriptional activity of the KAHRP gene. These studies suggest a functional role for this structural element in the developmental regulation of a P. falciparum erythrocytic gene.

Animals↗

Transcription mapping of a 100 kb locus of Plasmodium falciparum identifies an intergenic region in which transcription terminates and reinitiates.

We have mapped Plasmodium falciparum erythrocytic stage transcription units on chromosome 10 in the vicinity of the gene encoding the glycophorin binding protein (GBP130) using yeast artificial chromosomes (YACs). Three erythrocytic stage transcription units are clustered in a 40 kb region. Two of these genes are closely linked, separated by less than 2 kb. Nuclear run-on data demonstrate that transcription of these two genes, though unidirectional, is monocistronic. Within this intergenic region are the sites at which transcription of the upstream gene terminates and the GBP130 gene initiates. These studies represent the first description of the minimal and necessary cis-acting elements for transcription termination and initiation in this protozoan parasite.

Amino Acid Sequence↗

Characterization of yeast artificial chromosomes from Plasmodium falciparum: construction of a stable, representative library and cloning of telomeric DNA fragments.

Molecular genetic studies of the human malaria parasite Plasmodium falciparum have been hampered in part due to difficulties in stably cloning and propagating parasite genomic DNA in bacteria. This is thought to be a result of the unusual A+T bias (>80%) in the parasite's DNA. Pulsed-field gel electrophoretic separation of P. falciparum chromosomes has shown that large chromosomal polymorphisms, resulting from the deletion of DNA from chromosome ends, frequently occur. Understanding the biological implications of this chromosomal polymorphism will require the analysis of large regions of genomic, and in particular telomeric, DNA. To overcome the limitations of cloning parasite DNA in bacteria, we have cloned genomic DNA from the P. falciparum strain FCR3 in yeast as artificial chromosomes. A pYAC4 library with an average insert size of approximately 100 kb was established and found to have a three to fourfold redundancy for single-copy genes. Unlike bacterial hosts, yeast stably maintain and propagate large tracts of parasite DNA. Long-range restriction enzyme mapping of YAC clones demonstrates that the cloned DNA is contiguous and identical to the native parasite genomic DNA. Since the telomeric ends of chromosomes are underrepresented in YAC libraries, we have enriched for these sequences by cloning P. falciparum telomeric DNA fragments (from 40 to 130 kb) as YACs by complementation in yeast.

Animals↗

Organization of the human and mouse low-affinity Fc gamma R genes: duplication and recombination.

Receptors for immunoglobulin G immune complexes (Fc gamma RII and Fc gamma RIII) are expressed on most hematopoietic cells and show much structural and functional diversity. In order to determine the genetic basis for this diversity, a family of genes encoding the human and mouse receptors was isolated and characterized. Humans have five distinct genes for low-affinity Fc gamma Rs, in contrast to two in the mouse. With the use of yeast artificial chromosomes, the genes encoding the human receptors were oriented and linked, which established the structure of this complex locus. Comparison of the human and mouse genes generated a model for the evolutionary amplification of this locus.

Animals↗

A and T homopolymeric stretches mediate a DNA inversion in Plasmodium falciparum which results in loss of gene expression.

Ring-infected erythrocyte surface antigen-negative isolates of Plasmodium falciparum demonstrate a complex DNA rearrangement with inversion of 5' coding sequences, deletion of upstream and flanking sequences, and healing of the truncated chromosome by telomere addition. An inversion intermediate that results in the telomeric gene structure for RESA has been identified in the pathway. This inversion creates a mitotically stable substrate for the sequence-specific addition of telomere repeats at the deletion breakpoint.

Animals↗