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PubMed2006

A systems biology approach to multicellular and multi-generational radiation responses.

Recent studies have highlighted crosstalk between irradiated cells and non-irradiated bystander cells and have uncovered high-frequency phenotypes of genomic instability in the progeny of irradiated cells that cannot be solely explained by radiation-induced mutation. It is difficult to explain these multicellular and multi-generational phenomena using the current paradigm of radiation biology. Radiation-induced bystander effect is a type of multicellular response to radiation that illustrates that the unit of function in multicellular organisms is neither the genome nor the cell. Cell function in complex three-dimensional tissues is coordinated by soluble signaling peptides and by small molecules within the context of insoluble scaffolding provided by the extracellular matrix. Adaptive response and radiation-induced genomic instability could thus result from persistent signaling perturbations following radiation exposures. A model of radiation response based on the systems biology principles of network interconnectivity and spatial organization should reconcile the apparent contradiction of these cellular phenotypes within the higher order structure of tissues and organisms.

Animals↗

PubMed2004

Chromosome loops arising from intrachromosomal tethering of telomeres occur at high frequency in G1 (non-cycling) mitotic cells: Implications for telomere capture.

BACKGROUND: To investigate potential mechanisms for telomere capture the spatial arrangement of telomeres and chromosomes was examined in G1 (non-cycling) mitotic cells with diploid or triploid genomes. This was examined firstly by directly labelling the respective short arm (p) and long arm subtelomeres (q) with different fluorophores and probing cell preparations using a number of subtelomere probe pairs, those for chromosomes 1, 3, 4, 5, 6, 7, 9, 10, 12, 17, 18, and 20. In addition some interstitial probes (CEN15, PML and SNRPN on chromosome 15) and whole chromosome paint probes (e.g. WCP12) were jointly hybridised to investigate the co-localization of interphase chromosome domains and tethered subtelomeres. Cells were prepared by omitting exposure to colcemid and hypotonic treatments. RESULTS: In these cells a specific interphase chromosome topology was detected. It was shown that the p and q telomeres of the each chromosome associate frequently (80% pairing) in an intrachromosomal manner, i.e. looped chromosomes with homologues usually widely spaced within the nucleus. This p-q tethering of the telomeres from the one chromosome was observed with large (chromosomes 3, 4, 5), medium sized (6, 7, 9, 10, 12), or small chromosomes (17, 18, 20). When triploid nuclei were probed there were three tetherings of p-q subtelomere signals representing the three widely separated looped chromosome homologues. The separate subtelomere pairings were shown to coincide with separate chromosome domains as defined by the WCP and interstitial probes. The 20% of apparently unpaired subtelomeric signals in diploid nuclei were partially documented to be pairings with the telomeres of other chromosomes. CONCLUSIONS: A topology for telomeres was detected where looped chromosome homologues were present at G1 interphase. These homologues were spatially arranged with respect to one-another independently of other chromosomes, i.e. there was no chromosome order on different sides of the cell nuclei and no segregation into haploid sets was detected. The normal function of this high frequency of intrachromosomal loops is unknown but a potential role is likely in the genesis of telomere captures whether of the intrachromosomal type or between non-homologues. This intrachromosomal tethering of telomeres cannot be related to telomeric or subtelomeric sequences since these are shared in varying degree with other chromosomes. In our view, these intrachromosomal telomeric tetherings with the resulting looped chromosomes arranged in a regular topology must be important to normal cell function since non-cycling cells in G1 are far from quiescent, are in fact metabolically active, and these cells represent the majority status since only a small proportion of cells are normally dividing.

Journal Article↗

PubMed2006

A synchrotron FTIR microspectroscopy investigation of fungal hyphae grown under optimal and stressed conditions.

Synchrotron FTIR can provide high spatial resolution (<10 microm pixel size) in situ biochemical analyses of intact biotissues, an area of increasing importance in the post-genomic era, as gene functions and gene networks are coming under direct scrutiny. With this technique, we can simultaneously assess multiple aspects of cell biochemistry and cytoplasmic composition. In this paper, we report the first results of our synchrotron FTIR examination of hyphae of three important fungal model systems, each with sequenced genomes and a wealth of research: Aspergillus, Neurospora, and Rhizopus. We have analyzed the FTIR maps of Aspergillus nidulans cells containing the hypA1 allele, a well-characterized single-gene temperature-sensitive morphogenetic mutation. The hypA1 cells resemble wildtype at 28 degrees C but have growth defects at 42 degrees C. We have also investigated Neurospora and Rhizopus cultures grown in media with optimal or elevated pH. Significant differences between the spectra of the three fungi are likely related to differences in composition and structure. In addition, high spatial resolution synchrotron FTIR spectroscopy provides an outstanding method for monitoring subtle subcellular changes that accompany environmental stress.

Colony Count, Microbial↗

PubMed2004

Genome-wide identification of Arabidopsis coiled-coil proteins and establishment of the ARABI-COIL database.

Increasing evidence demonstrates the importance of long coiled-coil proteins for the spatial organization of cellular processes. Although several protein classes with long coiled-coil domains have been studied in animals and yeast, our knowledge about plant long coiled-coil proteins is very limited. The repeat nature of the coiled-coil sequence motif often prevents the simple identification of homologs of animal coiled-coil proteins by generic sequence similarity searches. As a consequence, counterparts of many animal proteins with long coiled-coil domains, like lamins, golgins, or microtubule organization center components, have not been identified yet in plants. Here, all Arabidopsis proteins predicted to contain long stretches of coiled-coil domains were identified by applying the algorithm MultiCoil to a genome-wide screen. A searchable protein database, ARABI-COIL (http://www.coiled-coil.org/arabidopsis), was established that integrates information on number, size, and position of predicted coiled-coil domains with subcellular localization signals, transmembrane domains, and available functional annotations. ARABI-COIL serves as a tool to sort and browse Arabidopsis long coiled-coil proteins to facilitate the identification and selection of candidate proteins of potential interest for specific research areas. Using the database, candidate proteins were identified for Arabidopsis membrane-bound, nuclear, and organellar long coiled-coil proteins.

Algorithms↗

PubMed2027

Identification of Genome-Wide Chromatin Structural Aberration in Cancer by Hi-C Analysis.

Aberrant three-dimensional genome organization is a hallmark of cancer, often driving oncogene activation through mechanisms such as enhancer hijacking. High-throughput chromosome conformation capture (Hi-C) maps these interactions on a genome-wide scale. Unlike earlier dilution-based methods, in situ Hi-C performs proximity ligation within intact nuclei, minimizing random ligation noise and enabling fine-scale structure detection. This chapter describes an optimized in situ Hi-C protocol tailored for cancer cell lines using MboI digestion and biotin-mediated pull-down to generate high-complexity libraries. We further outline a computational workflow that extends beyond standard topological mapping of compartments and topologically associating domains to identify cancer-specific aberrations. Specifically, we focus on detecting chromosomal rearrangements (structural variants) and characterizing the distinct circular topology of extrachromosomal DNA. This integrated experimental and analytical framework provides the necessary tools to dissect the spatial dysregulation underlying tumor evolution.

Humans↗

PubMed2002

Functional genomics of the social amoebae, Dictyostelium discoideum.

Dictyostelium discoideum is one of the simplest organisms to form a multicellular structure, and it offers several advantages as a model. In order to understand the genetic basis of the multicellular development, a comprehensive analysis of cDNAs is being performed. To date, about 75,000 ESTs have been collected at different stages of development. They have been assembled into about 6,400 independent sequences that represent 70-80% of all of the expected genes in D. discoideum. The results are available on the Internet. In addition to structural analyses, functional analyses of the temporal and spatial expression patterns and gene targeting are being carried out. Furthermore, there are plans to combine the information that is obtained from the cDNA, Genome, and Proteome Projects, as well as the published results, into an integrated database, DictyBase.

Animals↗

PubMed2005

The interphotoreceptor retinoid-binding protein (IRBP) of the chicken (Gallus gallus domesticus).

PURPOSE: Despite decades of investigation, the function of interphotoreceptor retinoid binding protein (IRBP), the most abundant protein in the interphotoreceptor matrix of vertebrates, remains enigmatic. Roles for IRBP in the visual cycle of rod photoreceptors and in the independent visual cycle of cone photoreceptors have been suggested, yet very little is known of the biology of IRBP in cone-dominant retinas, such as those of diurnal birds. Our aim was to identify and characterize expression of the IRBP of the cone-dominant chicken (Gallus gallus domesticus). METHODS: Chicken IRBP mRNA was identified by PCR cloning. Primary protein structure, genomic organization, and phylogenies were determined through comparative sequence analyses. Expression of IRBP mRNA was characterized by northern analysis and by in situ hybridization on cryosectioned chicken retina. Expression of the IRBP protein was characterized by western blotting and by indirect immunofluorescence on cryosectioned retina and on retinal whole mounts. RESULTS: The chicken IRBP gene encodes a secreted protein with a predicted 1,252 amino acid length. The gene structure for chicken IRBP resembles that of most other vertebrates, with four homologous, modular repeats and introns within only the fourth module. Each module is more homologous with the corresponding module in other species than it is with the remaining chicken modules. Chicken retinal tissue contains a single IRBP mRNA transcript of approximately 4.8 kb and western analysis of chicken retina shows a single major band of 140 kDa. Chicken IRBP mRNA is expressed exclusively by retinal photoreceptor cells and the intensity of the hybridization signal shows light/dark rhythmicity. The IRBP protein is localized to the interphotoreceptor matrix of the chicken retina and to intracellular regions of photoreceptors, with a spatial distribution indicating an association with cone outer segments. CONCLUSIONS: The high degree of conservation of IRBP's primary structure, genomic organization, and cell-specific expression within the retinas of all vertebrates examined to date, including those with cone-dominant retinas, implies a conserved role for IRBP in photoreceptor function and/or health. Expression of chicken IRBP and its mRNA are functionally regulated. This report provides a necessary first step to explore a specific function for IRBP in the cone visual cycle.

Amino Acid Sequence↗

PubMed1997

Functional Domains within the Nucleus of a Cell Infected with HSV-1.

HSV-1 is a nuclear replicating DNA virus capable of establishing both lytic and latent infections in mammalian cells. Expression of the more than 80 HSV genes (the majority of which do not contain introns) requires complex coordination of viral and cellular factors both temporally, at appropriate points during the infectious cycle, and spatially as the virus transcription, replication and DNA packaging factories develop in the cell nucleus. Whilst the HSV genome encodes sufficient proteins to sustain viral DNA replication, it is reliant upon its host cell for RNA polymerase II and RNA processing machinery, in addition to an increasing number of cellular cofactors, for gene expression. As HSV establishes a lytic infection, cellular gene expression and splicing are inhibited as cellular chromatin is displaced and a dramatic reorganisation of the host cell nucleus occurs. The formation of large protein-rich factories synthesising viral RNA and replicating and packaging the viral genomes is the most striking alteration. In addition to the synthetic factories, large clumps of cellular and viral intron-containing RNAs accumulate in the nucleus as a result of the inhibition of splicing, at locations which colocalise with splicing factors, but are separate from transcription sites. An essential HSV protein IE63, discussed here, has been identified with a role in the organisation of the nucleus at many levels including replication and transcription site formation, splicing factor organisation and the transport of RNA. This review is a summary of our present understanding of the organisation of the HSV infected cell nucleus, relating viral genomes, RNA, DNA and proteins in the context of the nucleus. However this is a rapidly evolving field and new factors (both viral and cellular) involved in the regulation of these functional domains are constantly being identified. Copyright 1997 by John Wiley & Sons, Ltd.

Journal Article↗

PubMed2002

Depletion of Mab21l1 and Mab21l2 messages in mouse embryo arrests axial turning, and impairs notochord and neural tube differentiation.

BACKGROUND: The nematode mab-21 gene specifies sensory ray cell identity and was first isolated because of its mutant sensory ray defects. Vertebrate Mab21 orthologs have since been identified in mammals and amphibians. In this report, we characterized in detail two Mab21 orthologs in mouse, Mab21l1 and Mab21l2. METHODS: We examined the genomic organizations of Mab21 genes and used northern blot and in situ hybridizations to assay their temporal-spatial expression pattern. Their embryonic functions were revealed by specific attenuation of Mab21 messages with antisense oligos in cultured embryos. RESULTS: Mab21l1 and Mab21l2 have very similar protein make-up and gene structures. Both genes were expressed in overlapping domains of actively differentiating embryonic tissues. In addition, Mab21l1 had unique expression in the lens vesicles and genital tubercle whereas Mab21l2 was expressed in the retinal epithelium and umbilical cord. Mab21l1 and Mab21l2 depleted embryos had severe defects in notochord, neural tube, organogenesis, vasculogenesis, and axial turning. CONCLUSIONS: The findings demonstrate that both Mab21 genes are required in developing embryos for embryonic turning, formation of the notochord, neural tube, and other organ tissues.

Animals↗

PubMedDate not supplied

Gene expression profiling of human diseases by serial analysis of gene expression.

Until recently, the approach to understanding the molecular basis of complex syndromes such as cancer, coronary artery disease, and diabetes was to study the behavior of individual genes. However, it is generally recognized that expression of a number of genes is coordinated both spatially and temporally and that this coordination changes during the development and progression of diseases. Newly developed functional genomic approaches, such as serial analysis of gene expression (SAGE) and DNA microarrays have enabled researchers to determine the expression pattern of thousands of genes simultaneously. One attractive feature of SAGE compared to microarrays is its ability to quantify gene expression without prior sequence information or information about genes that are thought to be expressed. SAGE has been successfully applied to the gene expression profiling of a number of human diseases. In this review, we will first discuss SAGE technique and contrast it to microarray. We will then highlight new biological insights that have emerged from its application to the study of human diseases.

Gene Expression↗

PubMed1996

ARPP-21: murine gene structure and promoter identification of a neuronal phosphoprotein enriched in the limbic striatum.

ARPP-21 (cAMP-regulated phosphoprotein, Mr = 21,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis) is a phosphoprotein highly enriched in concentration in the neurons of the limbic striatum. It is likely a third messenger in the intracellular cascade of events following neuronal stimulation by first-messenger activators of the adenylate cyclase system, including dopamine via the D1 receptor. ARPP-21 expression is restricted to telencephalic post-mitotic, post-migrational neurons, and its precise pattern of temporal and spatial expression makes it an attractive candidate for the study of transcriptional regulation of neuronal maturation. To define genomic regions likely to contain functional promoter elements, we isolated the murine ARPP-21 gene. Primer extension and T2 RNase protection analyses identified multiple transcription start sites, but 1.3 kb of 5'-flanking DNA revealed few consensus transcription factor binding sequences. A series of transient transfection assays in clonal cell lines which do not express ARPP-21 identified a basal promoter active in both neuronal and non-neuronal lines. Expression in all lines was decreased by the inclusion of regions further upstream, and extinguished by the inclusion of the first intron. Further analyses are likely to reveal cell specific regulatory sequences.

Animals↗

PubMed1994

Conformational behaviour of the active and inactive forms of the nucleocapsid NCp7 of HIV-1 studied by 1H NMR.

The nucleocapsid protein NCp7 of the human immunodeficiency virus type I (HIV-1) is a 72 amino acid peptide containing two zinc fingers of the type CX2CX4HX4C linked by a short basic sequence 29RAPRKKG35. NCp7 was shown to activate in vitro both viral RNA dimerization and replication primer tRNA(Lys,3) annealing to the initiation site of reverse transcription. In order to clarify the possible structural role of the zinc fingers in the various functions of NCp7, complete sequence specific 1H NMR assignment of the entire protein was achieved by two-dimensional NMR experiments. Moreover, to characterize the role of the peptide linker in NCp7 folding, a synthetic analogue with an inversion of Pro31 configuration was studied by NMR and fluorescence techniques. Several long range NOEs implying amino acid protons from the folded zinc fingers and the spacer, such as Ala25 and Trp37, Phe16 and Trp37, Arg32 and Trp37, Lys33 and Trp37, Cys18 and Lys33 disappeared in the D-Pro31 (12-53)NCp7, confirming the spatial proximity of the two CCHC boxes observed in the (13-51)NCp7. This was also confirmed by iodide fluorescence quenching experiments. The N and C-terminal parts of NCp7 displayed a large flexibility except for two short sequences Tyr56 to Gly58 and Tyr64 to Gly66, which seemed to oscillate between random-coil and helical conformations. The biological relevance of the structural characteristics of NCp7 was studied in vitro and in vivo. Substitution of Pro31 by D-Pro31 in the active (13-64)NCp7 peptide led to a severe reduction of dimerization in vitro. Moreover, site-directed mutagenesis substituting Leu for Pro31 resulted in the formation of non-infectious and immature viral particles. These results suggest that the spatial proximity of the zinc fingers induced by the peptide linker, plays a critical role in encapsidation of genomic RNA and morphogenesis of HIV-1 infectious particles.

Amino Acid Sequence↗

PubMed1996

The chromosomal distribution of mouse odorant receptor genes.

Odorant receptors (ORs) on nasal olfactory sensory neurons are encoded by a large multigene family. Each member of the family is expressed in a small percentage of neurons that are confined to one of several spatial zones in the nose but are randomly distributed throughout that zone. This pattern of expression suggests that when the sensory neuron selects which OR gene to express it may be confined to a particular zonal gene set of several hundred OR genes but select from among the members of that set via a stochastic mechanism. Both locus-dependent and locus-independent models of OR gene choice have been proposed. To investigate the feasibility of these models, we determined the chromosomal locations of 21 OR genes expressed in four different spatial zones. We found that OR genes are clustered within multiple loci that are broadly distributed in the genome. These loci lie within paralogous chromosomal regions that appear to have arisen by duplications of large chromosomal domains followed by extensive gene duplication and divergence. Our studies show that OR genes expressed in the same zone map to numerous loci; moreover, a single locus can contain genes expressed in different zones. These findings raise the possibility that OR gene choice may be locus-independent or involve consecutive stochastic choices.

Animals↗

PubMed2000

Vitamin A in pregnancy: requirements and safety limits.

Most of the functions of vitamin A are mediated through the binding of retinoic acid to specific nuclear receptors that regulate genomic expression. Recent experimental work in transgenic mice showed clearly that normal embryonic development depends on the correct spatial and temporal expression of the receptors in the differentiating cells and on the binding of specific forms of retinoic acid. This implies that the parent compound, vitamin A, is available in adequate forms and quantities. Excessive dietary intake of vitamin A has been associated with teratogenicity in humans in <20 reported cases over 30 y. However, caution must be exercised to avoid unnecessary supplementation of women of childbearing age. Hypovitaminosis A affects millions of women and children worldwide. The main consequence of a poor vitamin A supply during pregnancy is a low vitamin A status at birth and in the next few months. Vitamin A deficiency is strongly associated with depressed immune function and higher morbidity and mortality due to infectious diseases such as diarrhea, measles, and respiratory infections. Vitamin A deficiency is often associated with an increased mother-to-child transmission of HIV-1. The initiation of vitamin A supplementation should be carefully examined in each case according to the risk-to-benefit ratio. The final decision should take into account the estimated vitamin A status of the woman, the availability of vitamin A-rich foods in her diet, and whether supplementation can be supervised.

Animals↗

PubMed1990

Hierarchical analysis of population genetic variation in mitochondrial and nuclear genes of Daphnia pulex.

The geographic structure of Daphnia pulex populations from the central United States is analyzed with respect to isozyme and mitochondrial DNA variation. The species complex consists of cyclic and obligate parthenogens. A hierarchical analysis of population structure in the cyclic parthenogens by using a fixation-index approach indicates that this is one of the most extremely subdivided species yet studied. This genetic structure, much of which accrues within 100 km, is certainly due in part to the limited dispersal ability of Daphnia. However, previous work has shown that fluctuating selection can account for the spatial heterogeneity in isozyme frequencies in these populations. This may explain why the population subdivision for the mitochondrial genome increases approximately three times as rapidly with distance as does that for nuclear genes, which is slower than the neutral expectation. The obligate parthenogens are shown to be polyphyletic in origin, evolutionarily young, and, in some cases, geographically widespread.

Animals↗

PubMed1996

The fission yeast dma1 gene is a component of the spindle assembly checkpoint, required to prevent septum formation and premature exit from mitosis if spindle function is compromised.

Premature initiation of cytokinesis can lead to loss of chromosomes, and 'cutting' of the nucleus. Therefore, the proper spatial and temporal co-ordination of mitosis and cytokinesis is essential for maintaining the integrity of the genome. The fission yeast cdc16 gene is implicated both in the spindle assembly checkpoint and control of septum formation. To identify other proteins involved in these controls, we have isolated multicopy suppressors of the cdc16-116 mutation, and the characterization of one of these, dma1 (defective in mitotic arrest), is presented here. dma1 is not an essential gene, but in a dma1 null background (dma1-D1) the function of the spindle assembly checkpoint is compromised. If assembly of the spindle is prevented, dma1-D1 cells do not arrest, the activity of cdc2 kinase decays and cells form a division septum without completing a normal mitosis. dma1-D1 cells also show an increased rate of chromosome loss during exponential growth. Upon ectopic expression from an inducible promoter, dma1p delays progress through mitosis and inhibits septum formation, giving rise to elongated, multinucleate cells. We propose that dma1 is a component of the spindle assembly checkpoint, required to prevent septum formation and premature exit from mitosis if spindle function is impaired.

Amino Acid Sequence↗

PubMed2006

Surface manipulation of biomolecules for cell microarray applications.

Many biological events, such as cellular communication, antigen recognition, tissue repair and DNA linear transfer, are intimately associated with biomolecule interactions at the solid-liquid interface. To facilitate the study and use of these biological events for biodevice and biomaterial applications, a sound understanding of how biomolecules behave at interfaces and a concomitant ability to manipulate biomolecules spatially and temporally at surfaces is required. This is particularly true for cell microarray applications, where a range of biological processes must be duly controlled to maximize the efficiency and throughput of these devices. Of particular interest are transfected-cell microarrays (TCMs), which significantly widen the scope of microarray genomic analysis by enabling the high-throughput analysis of gene function within living cells. This article reviews this current research focus, discussing fundamental and applied research into the spatial and temporal surface manipulation of DNA, proteins and other biomolecules and the implications of this work for TCMs.

Cells, Immobilized↗

PubMed2026

Identification of cryosensitive niches and a targetable FOS/AP‑1 program in the human ovarian cortex by single‑cell and spatial transcriptomics.

BACKGROUND: The ovary is a vital and dynamic reproductive organ. Ovarian tissue cryopreservation (OTC) plays a vital role in preserving female fertility. However, the cellular subtypes most susceptible to cryoinjury and the molecular mechanisms underlying cryopreservation-associated damage remain poorly understood. This study aimed to identify cell populations vulnerable to freezing-thawing and to elucidate the key transcriptomic alterations and signaling pathways associated with ovarian cryoinjury at the single-cell and spatial levels. METHODS: Ovarian cortical tissues from patients undergoing three gender reassignment surgery (GRS) were divided into fresh and vitrification-rapid warming groups. Following collagenase IV digestion, 10x Genomics single-cell RNA-seq was used for dissociated ovarian cell suspensions (27,185 fresh and 25,480 frozen-thawed cells). Eight major cell clusters were identified. Additionally, 110 oocytes (66 fresh, 44 vitrification-rapid warming) were isolated and analyzed using the Smart-seq2 platform. Spatial transcriptomics was performed via BGI Stereo-seq. Molecular validation was performed via β-galactosidase staining, immunofluorescence, and qRT-PCR. RESULTS: Cryopreservation significantly altered the activity of pathways related to focal adhesion, oxidative stress, and apoptosis, particularly in stromal and perivascular cells. The number of FOS-positive perivascular cells was notably increased after vitrification-rapid warming, whereas the number of PTGDS-positive stromal cells decreased. Oocyte analysis revealed that cryopreservation primarily disrupted pathways involved in the cell cycle and meiosis, although the damage was not irreversible, supporting the relative safety of long-term cryostorage. Spatial transcriptomics and functional validation further confirmed the rapid and robust activation of the FOS/AP-1 pathway after vitrification-rapid warming, particularly in perivascular and granulosa cells. Treatment with T-5224 (a FOS/AP-1 inhibitor) significantly rescued the morphology and function of cultured frozen-thawed ovaries. CONCLUSIONS: Stromal and perivascular cells are the main cell types that are sensitive to ovarian cryopreservation. The FOS/AP-1 pathway is markedly activated after, suggesting the exacerbation of metabolic impairment. In oocytes within the ovarian cortex, the cell cycle and meiosis-related physiological processes were the primary processes affected.

Female↗