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At least 523 records · Page 29Linked to original sources
PubMed1998

Nuclear neighbours: the spatial and functional organization of genes and nuclear domains.

It is becoming clear that the cell nucleus is not only organized in domains but that these domains are also organized relative to each other and to the genome. Specific nuclear domains, enriched in different proteins and RNAs, are often found next to each other and next to specific gene loci. Several lines of investigation suggest that nuclear domains are involved in facilitating or regulating gene expression. The emerging view is that the spatial relationship between different domains and genes on different chromosomes, as found in the nucleolus, is a common organizational principle in the nucleus, to allow an efficient and controlled synthesis and processing of a range of gene transcripts.

Animals↗
PubMed2006

Stable and efficient intraocular gene transfer using pseudotyped EIAV lentiviral vectors.

BACKGROUND: We have developed minimal non-primate lentiviral vectors based on the equine infectious anaemia virus (EIAV). We evaluated the in vivo expression profiles of these vectors delivered regionally to ocular tissues to define their potential utility in ocular gene therapy. METHODS: EIAV vectors pseudotyped with VSV-G or rabies-G envelope proteins were delivered subretinally, intravitreally or into the anterior chambers (intracameral administration) in mice. Reporter gene (eGFP) expression was analysed using in vivo retinal imaging or histological examination of eyes and brains at intervals between 3 days and 16 months. We investigated the effects of vector titre, pseudotype, genome configuration, site of intraocular administration, intentional retinal trauma and the degree of retinal maturation on the spatial and temporal expression profiles of these vectors. RESULTS: Subretinal vector delivery resulted in efficient and stable transduction of retinal pigment epithelial (RPE) cells and variable transduction of photoreceptors up to 16 months post-injection. Retinal trauma facilitated the local transduction of neurosensory retinal cells. Intracameral administration of VSV-G- but not rabies-G-pseudotyped vectors produced stable eGFP expression in corneal endothelial cells and trabecular meshwork. CONCLUSIONS: The cellular tropism and expression kinetics of optimised EIAV vectors after intraocular administration make them attractive vehicles for delivering therapeutic genes in the management of inherited and acquired retinal and anterior segment disorders.

Animals↗
PubMed1991

Preservation of specific RNA distribution within the chromatin-depleted nuclear substructure demonstrated by in situ hybridization coupled with biochemical fractionation.

Biochemical fractionation procedures previously shown to remove 95% of cellular protein, DNA, and phospholipid, were combined with fluorescence in situ hybridization to provide a critical evaluation of the retention and spatial preservation of specific primary transcripts within the chromatin-depleted nuclear substructure, operationally defined as the nuclear "matrix." This unique approach made it possible to directly address whether nuclear extraction procedures preserve, create, or destroy ribonucleoprotein filament structures. Comparison of nuclei before and after fractionation demonstrated that localized foci or "tracks" of specific nRNA are unambiguously retained in the nuclear matrix preparation. Two well-characterized nuclear fractionation procedures were used and three Epstein-Barr virus-infected cell types investigated, including latently and permissively infected cells carrying integrated or episomal genomes. The EBV primary transcripts as well as nucleolar RNA were preserved within the remaining nuclear substructure with unambiguous spatial and quantitative fidelity. Image processing and quantitative microfluorimetry, together with [3H]thymidine labeling of DNA, show that essentially 100% of the RNA signal intensity remained after removal of 85% of the DNA. That the native RNA distribution was unchanged was shown in other experiments in which the same individual nRNA tracks were examined before and after fractionation. Results conclusively demonstrate that the tight restriction of RNA to highly localized sites is independent of bulk DNA removal and of extensive extraction of proteins and phospholipids. Hence, this work provides direct visual evidence that the primary transcripts studied are localized via their binding to, or comprising part of, non-chromatin nuclear substructure.

Cell Line↗
PubMed2026

Spatial habitat radiomics predicts tertiary lymphoid structure status and identifies an IDO1+ migratory dendritic cell axis in breast cancer.

BACKGROUND: Tertiary lymphoid structures (TLS) are spatially organized immune niches associated with therapeutic response and favorable outcomes in breast cancer (BC). However, TLS assessment currently relies on invasive tissue-based analyses, and the biological mechanisms underlying imaging-based TLS prediction remain poorly understood. METHODS: We developed and validated a spatial heterogeneity-based radiomic TLS signature (shTLS) using dynamic contrast-enhanced MRI to non-invasively predict TLS status across multicenter BC cohorts. Spatial habitat radiomics were used to capture intratumoral and peritumoral immune-related heterogeneity. Integrated multi-omics analyses, including transcriptomics, pathomics, genomics, single-cell RNA sequencing, immunohistochemistry, and multiplex immunofluorescence, were performed to biologically interpret shTLS-defined subgroups. Functional drug-sensitivity assays were conducted to assess therapeutic implications. RESULTS: The shTLS model achieved robust predictive performance across independent cohorts and molecular subtypes. High shTLS scores were associated with immune-inflamed tumors characterized by spatially clustered activated T cells and dendritic cells (DCs). In contrast, shTLS-low tumors exhibited an immunosuppressive spatial niche with peripheral accumulation of CD4+ PD-1+ T cells and plasma cells, increased immune-tumor separation, and enhanced inflammatory and immunoregulatory signaling. An indoleamine 2,3-dioxygenase 1 (IDO1)-associated immunoregulatory program was observed in the shTLS-low tumors, which appeared to be preferentially expressed by LAMP3+CCR7+ migratory DCs. Pharmacologic inhibition of IDO1 enhanced chemotherapy and CDK4/6 inhibitor sensitivity in vitro. CONCLUSION: This study establishes spatial radiomics as a non-invasive approach to decode TLS-associated immune ecosystems and supports the presence of an IDO1-associated immunosuppressive phenotype, providing biological insight and translational rationale for patient stratification and future combination strategies.

Humans↗
PubMed2001

Use of differential fluorescence induction and optical trapping to isolate environmentally induced genes.

The techniques of differential fluorescence induction (DFI) and optical trapping (OT) have been combined to allow the identification of environmentally induced genes in single bacterial cells. Designated DFI-OT, this technique allows the in situ isolation of genes driving the expression of green fluorescent protein (Gfp) using temporal and spatial criteria. A series of plasmid-based promoter probe vectors (pOT) was developed for the construction of random genomic libraries that are linked to gfpUV or egfp. Bacteria that do not express Gfp on laboratory medium (i.e. non-fluorescent) were inoculated into the environment, and induced genes were detected with a combined fluorescence/optical trapping microscope. Using this selection strategy, rhizosphere-induced genes with homology to thiamine pyrophosphorylase (thiE) and cyclic glucan synthase (ndvB) were isolated. Other genes were expressed late in the stationary phase or as a consequence of surface-dependent growth, including fixND and metX, and a putative ABC transporter of putrescine. This strategy provides a unique ability to combine spatial, temporal and physical information to identify environmental regulation of bacterial gene expression.

Bacterial Proteins↗
PubMed2004

Root growth maintenance during water deficits: physiology to functional genomics.

Progress in understanding the network of mechanisms involved in maize primary root growth maintenance under water deficits is reviewed. These include the adjustment of growth zone dimensions, turgor maintenance by osmotic adjustment, and enhanced cell wall loosening. The role of the hormone abscisic acid (ABA) in maintaining root growth under water deficits is also addressed. The research has taken advantage of kinematic analysis, i.e. characterization of spatial and temporal patterns of cell expansion within the root growth zone. This approach revealed different growth responses to water deficits and ABA deficiency in distinct regions of the root tip. In the apical 3 mm region, elongation is maintained at well-watered rates under severe water deficit, although only in ABA-sufficient roots, whereas the region from 3-7 mm from the apex exhibits maximum elongation in well-watered roots, but progressive inhibition of elongation in roots under water deficit. This knowledge has greatly facilitated discovery of the mechanisms involved in regulating the responses. The spatial resolution with which this system has been characterized and the physiological knowledge gained to date provide a unique and powerful underpinning for functional genomics studies. Characterization of water deficit-induced changes in transcript populations and cell wall protein profiles within the growth zone of the maize primary root is in progress. Initial results from EST and unigene analyses in the tips of well-watered and water-stressed roots highlight the strength of the kinematic approach to transcript profiling.

Adaptation, Physiological↗
PubMed2006

Definition and spatial annotation of the dynamic secretome during early kidney development.

The term "secretome" has been defined as a set of secreted proteins (Grimmond et al. [2003] Genome Res 13:1350-1359). The term "secreted protein" encompasses all proteins exported from the cell including growth factors, extracellular proteinases, morphogens, and extracellular matrix molecules. Defining the genes encoding secreted proteins that change in expression during organogenesis, the dynamic secretome, is likely to point to key drivers of morphogenesis. Such secreted proteins are involved in the reciprocal interactions between the ureteric bud (UB) and the metanephric mesenchyme (MM) that occur during organogenesis of the metanephros. Some key metanephric secreted proteins have been identified, but many remain to be determined. In this study, microarray expression profiling of E10.5, E11.5, and E13.5 kidney and consensus bioinformatic analysis were used to define a dynamic secretome of early metanephric development. In situ hybridisation was used to confirm microarray results and clarify spatial expression patterns for these genes. Forty-one secreted factors were dynamically expressed between the E10.5 and E13.5 timeframe profiled, and 25 of these factors had not previously been implicated in kidney development. A text-based anatomical ontology was used to spatially annotate the expression pattern of these genes in cultured metanephric explants.

Animals↗
PubMed2003

Identification of an enhancer region in the mouse ephA8 locus directing expression to the anterior region of the dorsal mesencephalon.

Eph receptors and ephrins are dynamically expressed in a wide range of regions of the vertebrate during embryogenesis. The dorsal mesencephalon appears to be segmented into two broad regions demarcated by the mutually exclusive expression of EphA receptors and ephrinA ligands. It is of considerable interest to elucidate how these expression domains are established in the development of the mesencephalon. In this study, we used a transgenic approach to define the cis-acting DNA regulatory elements involved in the anterior mesencephalon-specific expression of the mouse ephA8 gene. Our analyses of the temporal and spatial expression patterns of various ephA8/lacZ gene fusions in transgenic mice revealed that the 10-kb genomic DNA 5' immediately upstream of the ephA8 coding sequence is capable of directing lacZ expression in an ephA8-specific manner. Further deletion analyses of the ephA8 genomic region led to the identification of a 1-kb enhancer region, which directs expression in the embryo to the anterior region of the developing midbrain. This ephA8-specific regulatory DNA sequences can now be used in biochemical analyses to identify proteins modulating the anterior differentiation of the optic tectum, and in functional analyses to direct the expression of other developmentally important genes to this region.

5' Flanking Region↗
PubMed2026

High-Density SNP Genotyping Reveals High Population Connectivity and Limited Spatial Genetic Structure in Apodemus flavicollis and Apodemus sylvaticus.

High-density SNP arrays are increasingly used in ecological and evolutionary studies, yet their application in wild species remains challenging. In this study, we evaluated the performance of the Affymetrix Axiom Mouse HD array, originally developed for Mus musculus, in two wild small mammals, Apodemus flavicollis and Apodemus sylvaticus, with particular focus on genetic diversity and population connectivity across seven sampling sites within a fragmented landscape. A total of 96 individuals (43 A. flavicollis and 53 A. sylvaticus) were genotyped using a 616K SNP array. After quality control filtering for missingness and minor allele frequency, more than 160,000 high-quality autosomal SNPs were retained for each species. Despite being designed for a different species, the array effectively discriminated between A. flavicollis and A. sylvaticus, with principal component analysis clearly separating the two species. Levels of genetic diversity were comparable across sites, with mean observed heterozygosity around 0.33 and consistently negative F IS values, indicating a slight excess of heterozygotes. Population structure analyses revealed extremely weak spatial genetic differentiation. ADMIXTURE supported a single genetic cluster (K = 1) within each species, while analysis of molecular variance attributed more than 99% of genetic variation to within-individual components. Pairwise relationship analyses showed that related individuals were not confined to single sites but occurred across sampling locations, supporting ongoing gene flow even across the fragmented landscape. No significant isolation-by-distance pattern was detected. Overall, our results indicate high population connectivity and limited spatial genetic structuring in both species across the study area, consistent with the documented dispersal capacity of these species at the spatial scale investigated. Moreover, this study demonstrates that high-density SNP arrays can provide powerful genomic tools for investigating dispersal dynamics and population structure in closely related wildlife species under habitat fragmentation, where subtle genetic patterns may otherwise remain undetected.

Apodemus species↗
PubMed2006

Homologous chromosome pairing in Schizosaccharomyces pombe.

Homologous chromosome pairing is a central feature of meiosis I, contributing to the correct segregation of chromosomes during meiosis. The fission yeast, Schizosaccharomyces pombe, has been widely used to study meiotic chromosome dynamics, partly because studies in this yeast are simplified due to the lack of post-pairing synaptic structures. Chromosome pairing in Sz. pombe occurs differentially throughout the genome. Telomeres cluster at the spindle pole body (SPB) at the onset of meiosis, imposing a spatial restriction on pairing events. Subsequently, centromeres dissociate from the SPB and pair in a recombination- and heterochromatin (Swi6)-independent fashion. Pairing of telomere distal regions occurs during meiotic prophase, concomitant with a dynamic association/dissociation of homologous regions, with interhomologue associations becoming increasingly stable. The stabilization of paired regions is enhanced by factors required for the initiation of meiotic recombination, suggesting that recombination stabilizes paired regions. However, substantial pairing is initiated in the absence of recombination; this is dependent upon another factor, the conserved Meu13 protein, demonstrating that recombination is not required for initial pairing interactions. During meiotic prophase Sz. pombe exhibits a pronounced dynein-dependent nuclear oscillation, which drives the pairing of centromeric and interstitial regions. Dynein is also required for the significant levels of achiasmate reductional segregation observed in Sz. pombe, possibly implicating the centromere-associated pairing with achiasmate homologue segregation. Whilst Sz. pombe does not form discernable synaptic structures continuously along the meiotic chromosomes, it does form proteinacious, meiosis-specific, linear structures (linear elements). However, the role, if any, of these structures in mediating homologue pairing is unknown.

Cell Cycle Proteins↗
PubMed2007

Improved embryonic stem cell technologies.

Murine embryonic stem (ES) cells have become an indispensable tool for investigating genetic function both in vitro and, importantly, in vivo. Recent advances, including tetraploid aggregation, new site-specific recombinases and RNAi, have enabled more sophisticated manipulation of the ES cell genome. For instance, it is now possible to control gene expression in both a temporally and spatially restricted manner. Such new technologies are answering complex questions surrounding the function and interaction of an increasing number of genes. This chapter will review both the history and recent technological progress that has been made in mouse ES cell derivation, genetic manipulation and the generation of ES cell-derived chimaeric animals.

Animals↗
PubMed1998

Topoisomerase I of Helicobacter pylori: juxtaposition with a flagellin gene (flaB) and functional requirement of a fourth zinc finger motif.

Cloning and nucleotide sequence analysis showed that in Helicobacter pylori the gene encoding topoisomerase I (topA) lies about 170 nucleotides upstream from flaB, a gene encoding one of the two flagellin proteins that is required for virulence. The topA and flaB genes are divergently transcribed. The orientation and spatial relationship between flaB and topA are remarkably conserved among strains of a bacterium in which genomic rearrangements are common. The deduced amino acid sequence of topoisomerase I revealed four zinc finger motifs, one more than has been reported previously for the Escherichia coli homologue. The additional motif, which is near the C-terminus of the protein, appears to be essential for function since mutations in that region are lethal. These data show that TopA proteins can be divided into several classes on the basis of zinc finger motifs and raise the interesting possibility that the H. pylori enzyme has local topological effects focussed on a flagellin gene.

Bacterial Proteins↗
PubMed2004

Investigation of different transcript quantitation tools for high-throughput mapping of brain gene expression using voxelation.

Voxelation is a new approach for genome scale acquisition of brain gene expression patterns. The method employs high-throughput analysis of spatially registered voxels (cubes) to create multiple volumetric images of brain gene expression, similar to those obtained from biomedical imaging systems. The spatial resolution of voxelation depends on voxel size, with smaller voxels giving higher resolution. An important question is the applicability of different transcript profiling tools for the various levels of resolution that can be employed. Here, we describe the use of three methods to analyze voxel transcript abundance: real-time PCR, microarray analysis and linear amplification coupled with microarrays. We show statistically significant concordance between real-time PCR and microarray analysis for the myelin basic protein gene in human brain specimens at differing levels of spatial resolution. In addition, we also demonstrate the feasibility of using linear amplification coupled with microarray analysis to create voxelation maps from the mouse brain at high resolution, 1 microl. These data indicate the suitability of a number of transcript profiling tools for various levels of spatial resolution in voxelation.

Brain↗
PubMed1996

Population genetics with RAPD-PCR markers: the breeding structure of Aedes aegypti in Puerto Rico.

RAPD-PCR polymorphisms at 57 presumptive loci were used to examine the breeding structure of the mosquito Aedes aegypti in Puerto Rico. Mosquitoes were sampled from 16 locations in six cities and samples were located in a nested spatial design to examine local patterns of gene flow. Allele frequencies were estimated assuming (1) that genomic regions amplified by RAPD-PCR segregate as dominant alleles, (2) that genotypes at RAPD loci are in Hardy-Weinberg proportions, (3) identity in state (iis) among dominant amplified alleles and (4) iis among null alleles. The average genic heterozygosity was 0.354, more than twice the level detected in earlier allozyme surveys. Nested analysis of variance indicated extensive genetic differentiation among locations within cities. Effective migration rates (Nm) among cities were estimated from FST assuming an island model of migration. Estimates of Nm ranged from 9.7 to 12.2 indicating a high dispersal rate. The large number of polymorphisms revealed by RAPD-PCR allowed the distribution of FST and linkage disequilibrium to be examined among loci and demonstrated that small samples inflate FST and linkage disequilibrium. No linkage disequilibrium maintained through epistasis was detected among alleles at the 57 loci.

Aedes↗
PubMed1988

Self-incompatibility genes of Brassica oleracea: Expression, isolation, and structure.

We demonstrate by in situ hybridization the cell-type-specific expression of transcripts encoded by the self-incompatibility (S) locus of Brassica oleracea. These transcripts are not detected early in stigma development, their expression is switched on in the papillar cells of the stigma surface, and their levels increase in these cells in correlation with the acquisition by the stigma of the self-incompatibility response. By using a probe derived from the untranslated sequences at the 3' end of S cDNA, an S-gene copy expressed in the papillar cells has been isolated from among the multiple S-related copies that occur in the Brassica genome. Structural analysis of this gene shows that it lacks introns. In light of the strict spatial and temporal regulation of S-gene expression in precisely the cells that constitute the barrier to self-pollination, the self-incompatibility response may be viewed as a cell-cell interaction between one pollen grain and one papillar cell.

Journal Article↗
PubMed2002

Identification of a novel cis-regulatory element involved in the heat shock response in Caenorhabditis elegans using microarray gene expression and computational methods.

We report here the identification of a previously unknown transcription regulatory element for heat shock (HS) genes in Caenorhabditis elegans. We monitored the expression pattern of 11,917 genes from C. elegans to determine the genes that were up-regulated on HS. Twenty eight genes were observed to be consistently up-regulated in several different repetitions of the experiments. We analyzed the upstream regions of these genes using computational DNA pattern recognition methods. Two potential cis-regulatory motifs were identified in this way. One of these motifs (TTCTAGAA) was the DNA binding motif for the heat shock factor (HSF), whereas the other (GGGTGTC) was previously unreported in the literature. We determined the significance of these motifs for the HS genes using different statistical tests and parameters. Comparative sequence analysis of orthologous HS genes from C. elegans and Caenorhabditis briggsae indicated that the identified DNA regulatory motifs are conserved across related species. The role of the identified DNA sites in regulation of HS genes was tested by in vitro mutagenesis of a green fluorescent protein (GFP) reporter transgene driven by the C. elegans hsp-16-2 promoter. DNA sites corresponding to both motifs are shown to play a significant role in up-regulation of the hsp-16-2 gene on HS. This is one of the rare instances in which a novel regulatory element, identified using computational methods, is shown to be biologically active. The contributions of individual sites toward induction of transcription on HS are nonadditive, which indicates interaction and cross-talk between the sites, possibly through the transcription factors (TFs) binding to these sites.

Animals↗
PubMed1991

Evolution in absence of mutation.

Each part of a living organism contains a concealed totality which can eventually express itself partially or totally. The extent of the expression is spatially and temporally defined by the morphogenetic field and the environment. Neither the field nor the genome are evolving units. Evolution is produced by the boundary conditions and results in progressive losses, to which the organisms responds. Genetic losses can stabilize the new balance. A living system can be fruitfully compared to a semiotic system.

Animals↗
PubMed2003

Polar localization of replicon origins in the multipartite genomes of Agrobacterium tumefaciens and Sinorhizobium meliloti.

The origins of replication of many different bacteria have been shown to reside at specific subcellular locations, but the mechanisms underlying their positioning and segregation are still being elucidated. In particular, little is known about the replication of multipartite genomes in bacteria. We determined the cellular positions of the origins of the replicons in the alpha proteobacteria Agrobacterium tumefaciens and Sinorhizobium meliloti and found that they are located at the poles of the cells. Our work demonstrates the conserved extreme polar localization of circular chromosome origins in these alpha proteobacteria and is also the first to specify the cellular location of origin regions from the repABC family. The cellular location of a derivative of the RK2 plasmid is distinct from that of the alpha proteobacterium genomic replicon origins but is conserved across bacteria. Colocalization experiments with the genomic replicons of A. tumefaciens revealed that the repABC replicons, although preferentially positioned at the cell pole, colocalize only rarely. For the repABC replicons in this organism, occupying discrete spatial locations may contribute to their coexistence and stable inheritance.

Agrobacterium tumefaciens↗