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PubMed2004

Genomic structure and promoter characterization of the human Sprouty4 gene, a novel regulator of lung morphogenesis.

The expression of Sprouty4 (Spry4), an intracellular FGF receptor antagonist, shows a temporally and spatially restricted pattern in embryonic lung and is induced by ERK signaling. To clarify the molecular mechanisms regulating Spry4 transcription, the genomic structure of the human Sprouty4 (hSpry4) gene was first determined by using the GenomeWalker kit. The hSpry4 gene spans > 14 kb and is organized in three exons and two introns. Multiple transcription start sites were subsequently mapped by 5'-rapid amplification of cDNA ends. Analysis of up to 4 kb of sequence in the 5'-flanking region of the gene showed the presence of multiple potential transcription factor binding sites but no TATA or CAAT boxes. Transient transfection using luciferase reporter gene constructs with progressive deletions of the hSpry4 5'-flanking region revealed that the core promoter activity is located within the proximal 0.4-kb region, whereas the minimal ERK-inducible promoter activity is between -69 and -31. Homology analysis further showed that the core promoter region of the hSpry4 gene exhibits significant similarity to the 5'-flanking region of the mouse gene.

Animals↗
PubMed2004

High resolution microarray comparative genomic hybridisation analysis using spotted oligonucleotides.

BACKGROUND: Currently, comparative genomic hybridisation array (array CGH) is the method of choice for studying genome wide DNA copy number changes. To date, either amplified representations of bacterial artificial chromosomes (BACs)/phage artificial chromosomes (PACs) or cDNAs have been spotted as probes. The production of BAC/PAC and cDNA arrays is time consuming and expensive. AIM: To evaluate the use of spotted 60 mer oligonucleotides (oligos) for array CGH. METHODS: The hybridisation of tumour cell lines with known chromosomal aberrations on to either BAC or oligoarrrays that are mapped to the human genome. RESULTS: Oligo CGH was able to detect amplifications with high accuracy and greater spatial resolution than other currently used array CGH platforms. In addition, single copy number changes could be detected with a resolution comparable to conventional CGH. CONCLUSIONS: Oligos are easy to handle and flexible, because they can be designed for any part of the genome without the need for laborious amplification procedures. The full genome array, containing around 30000 oligos of all genes in the human genome, will represent a big step forward in the analysis of chromosomal copy number changes. Finally, oligoarray CGH can easily be used for any organism with a fully sequenced genome.

Breast Neoplasms↗
PubMed2003

Comparative genomic hybridization. Synchronous occurrence of focal nodular hyperplasia and hepatocellular carcinoma in the same liver is not based on common chromosomal aberrations.

Occasionally hepatocellular carcinomas (HCCs) occur synchronously with or within focal nodular hyperplasias (FNHs), raising the question of a putative causal relationship. In the present study, we used comparative genomic hybridization to investigate the occurrence of genomic aberrations in FNHs, which might lead to hepatocarcinogenesis. Tissue samples from FNHs and nonlesional liver tissue were obtained from 7 women. None of the patients had a chronic diffuse liver disease. A synchronous HCC not spatially related to FNH was present in 1 patient. Two patients had received oral contraceptives. Genomic aberrations were found in only 1 FNH. No aberration was found in the FNH occurring synchronously with HCC, but the HCC included gains at chromosomes 1q, 5, 12, and 19q and losses at 4p, 7q22-q35, 9p, 17p, 21q, and 22q. No aberrations were found in nonneoplastic liver tissues. Our findings support the notion that FNH is not a preneoplastic lesion for the occurrence of HCC in humans and that the synchronous occurrence of FNH and HCC is coincidental in our case.

Adult↗
PubMed2005

Genetically encoded sensors for metabolites.

BACKGROUND: Metabolomics, i.e., the multiparallel analysis of metabolite changes occurring in a cell or an organism, has become feasible with the development of highly efficient mass spectroscopic technologies. Functional genomics as a standard tool helped to identify the function of many of the genes that encode important transporters and metabolic enzymes over the past few years. Advanced expression systems and analysis technologies made it possible to study the biochemical properties of the corresponding proteins in great detail. We begin to understand the biological functions of the gene products by systematic analysis of mutants using systematic PTGS/RNAi, knockout and TILLING approaches. However, one crucial set of data especially relevant in the case of multicellular organisms is lacking: the knowledge of the spatial and temporal profiles of metabolite levels at cellular and subcellular levels. METHODS: We therefore developed genetically encoded nanosensors for several metabolites to provide a basic set of tools for the determination of cytosolic and subcellular metabolite levels in real time by using fluorescence microscopy. RESULTS: Prototypes of these sensors were successfully used in vitro and also in vivo, i.e., to measure sugar levels in fungal and animal cells. CONCLUSIONS: One of the future goals will be to expand the set of sensors to a wider spectrum of substrates by using the natural spectrum of periplasmic binding proteins from bacteria and by computational design of proteins with altered binding pockets in conjunction with mutagenesis. This toolbox can then be applied for four-dimensional imaging of cells and tissues to elucidate the spatial and temporal distribution of metabolites as a discovery tool in functional genomics, as a tool for high-throughput, high-content screening for drugs, to test metabolic models, and to analyze the interplay of cells in a tissue or organ.

Animals↗
PubMed2006

Genetic organization and embryonic expression of the ParaHox genes in the sea urchin S. purpuratus: insights into the relationship between clustering and colinearity.

The ANTP family of homeodomain transcription factors consists of three major groups, the NKL, the extended Hox, and the Hox/ParaHox family. Hox genes and ParaHox genes are often linked in the genome forming two clusters of genes, the Hox cluster and the ParaHox cluster, and are expressed along the major body axis in a nested fashion, following the relative positions of the genes within these clusters, a property called colinearity. While the presences of a Hox cluster and a ParaHox cluster appear to be primitive for bilaterians, few taxa have actually been examined for spatial and temporal colinearity, and, aside from chordates, even fewer still manifest it. Here we show that the ParaHox genes of the sea urchin Strongylocentrotus purpuratus show both spatial and temporal colinearity, but with peculiarities. Specifically, two of the three ParaHox genes-discovered through the S. purpuratus genome project-Sp-lox and Sp-Cdx, are expressed in the developing gut with nested domains in a spatially colinear manner. However, transcripts of Sp-Gsx, although anterior of Sp-lox, are detected in the ectoderm and not in the gut. Strikingly, the expression of the three ParaHox genes would follow temporal colinearity if they were clustered in the same order as in chordates, but each ParaHox gene is actually found on a different genomic scaffold (>300 kb each), which suggests that they are not linked into a single coherent cluster. Therefore, ParaHox genes are dispersed in the genome and are used during embryogenesis in a temporally and spatially coherent manner, whereas the Hox genes, now fully sequenced and annotated, are still linked and are employed as a complex only during the emergence of the adult body plan in the larva.

Amino Acid Sequence↗
PubMed2006

angaGEDUCI: Anopheles gambiae gene expression database with integrated comparative algorithms for identifying conserved DNA motifs in promoter sequences.

BACKGROUND: The completed sequence of the Anopheles gambiae genome has enabled genome-wide analyses of gene expression and regulation in this principal vector of human malaria. These investigations have created a demand for efficient methods of cataloguing and analyzing the large quantities of data that have been produced. The organization of genome-wide data into one unified database makes possible the efficient identification of spatial and temporal patterns of gene expression, and by pairing these findings with comparative algorithms, may offer a tool to gain insight into the molecular mechanisms that regulate these expression patterns. DESCRIPTION: We provide a publicly-accessible database and integrated data-mining tool, angaGEDUCI, that unifies 1) stage- and tissue-specific microarray analyses of gene expression in An. gambiae at different developmental stages and temporal separations following a bloodmeal, 2) functional gene annotation, 3) genomic sequence data, and 4) promoter sequence comparison algorithms. The database can be used to study genes expressed in particular stages, tissues, and patterns of interest, and to identify conserved promoter sequence motifs that may play a role in the regulation of such expression. The database is accessible from the address http://www.angaged.bio.uci.edu. CONCLUSION: By combining gene expression, function, and sequence data with integrated sequence comparison algorithms, angaGEDUCI streamlines spatial and temporal pattern-finding and produces a straightforward means of developing predictions and designing experiments to assess how gene expression may be controlled at the molecular level.

Algorithms↗
PubMed2000

Hepatitis delta virus replication generates complexes of large hepatitis delta antigen and antigenomic RNA that affiliate with and alter nuclear domain 10.

Hepatitis delta virus (HDV), a single-stranded RNA virus, bears a single coding region whose product, the hepatitis delta antigen (HDAg), is expressed in two isoforms, small (S-HDAg) and large (L-HDAg). S-HDAg is required for replication of HDV, while L-HDAg inhibits viral replication and is required for the envelopment of the HDV genomic RNA by hepatitis B virus proteins. Here we have examined the spatial distribution of HDV RNA and proteins in infected nuclei, with particular reference to specific nuclear domains. We found that L-HDAg was aggregated in specific nuclear domains and that over half of these domains were localized beside nuclear domain 10 (ND10). At later times, ND10-associated proteins like PML were found in larger HDAg complexes that had developed into apparently hollow spheres. In these larger complexes, PML was found chiefly in the rims of the spheres, while the known ND10 components Sp100, Daxx, and NDP55 were found in the centers of the spheres. Thus, ND10 proteins that normally are closely linked separate within HDAg-associated complexes. Viral RNA of antigenomic polarity, whether expressed from genomic RNA or directly from introduced plasmids, colocalizes with L-HDAg and the transcriptional repressor PML. In contrast, HDV genomic RNA was distributed more uniformly throughout the nucleus. These results suggest that different host protein complexes may assemble on viral RNA strands of different polarities, and they also suggest that this RNA virus, like DNA viruses, can alter the distribution of ND10-associated proteins. The fact that viral components specifically linked to repression of replication can associate with one of the ND10-associated proteins (PML) raises the possibility that this host protein may play a role in the regulation of HDV RNA synthesis.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Spatial organization of large-scale chromatin domains in the nucleus: a magnified view of single chromosome territories.

We have analyzed the spatial organization of large scale chromatin domains in chinese hamster fibroblast, human lymphoid (IM-9), and marsupial kidney epithelial (PtK) cells by labeling DNA at defined stages of S phase via pulsed incorporation of halogenated deoxynucleosides. Most, if not all, chromosomes contribute multiple chromatin domains to both peripheral and internal nucleoplasmic compartments. The peripheral compartment contains predominantly late replicating G/Q bands, whereas early replicating R bands preferentially localize to the internal nucleoplasmic compartment. During mitosis, the labeled chromatin domains that were separated in interphase form a pattern of intercalated bands along the length of each metaphase chromosome. The transition from a banded (mitotic) to a compartmentalized (interphasic) organization of chromatin domains occurs during the late telophase/early G1 stage and is independent of transcriptional activation of the genome. Interestingly, generation of micronuclei with a few chromosomes showed that the spatial separation of early and late replicating chromatin compartments is recapitulated independently of chromosome number, even in micronuclei containing only a single chromosome. Our data strongly support the notion that the compartmentalization of large-scale (band size) chromatin domains seen in the intact nucleus is a magnified image of a similar compartmentalization occurring in individual chromosome territories.

Animals↗
PubMed1992

Reversible histone modifications and the chromosome cell cycle.

During the eukaryotic cell cycle, chromosomes undergo large structural transitions and spatial rearrangements that are associated with the major cell functions of genome replication, transcription and chromosome condensation to metaphase chromosomes. Eukaryotic cells have evolved cell cycle dependent processes that modulate histone:DNA interactions in chromosomes. These are; i) acetylations of lysines; ii) phosphorylations of serines and threonines and iii) ubiquitinations of lysines. All of these reversible modifications are contained in the well-defined very basic N- and C-terminal domains of histones. Acetylations and phosphorylations markedly affect the charge densities of these domains whereas ubiquitination adds a bulky globular protein, ubiquitin, to lysines in the C-terminal tails of H2A and H2B. Histone acetylations are strictly associated with genome replication and transcription; histone H1 and H3 phosphorylations correlate with the process of chromosome condensation. The subunits of histone H1 kinase have now been shown to be cyclins and the p34CDC2 kinase product of the cell cycle control gene CDC2. It is probable that all of the processes that control chromosome structure:function relationships are also involved in the control of the cell cycle.

Acetylation↗
PubMed1995

The Drosophila E93 gene from the 93F early puff displays stage- and tissue-specific regulation by 20-hydroxyecdysone.

Pulses of ecdysteroids induce dramatic changes in gene expression that direct the early stages of Drosophila metamorphosis. This gene activity is reflected by the appearance of early and late puffs in the salivary gland polytene chromosomes. Curiously, the early puff genes that have been studied to date are induced by both the late larval and prepupal pulses of ecdysteroids and are expressed in many ecdysteroid target tissues, raising the question of how the hormone directs the complex stage- and tissue-specific responses associated with metamorphosis. In an effort to address this question, we have isolated and characterized the E93 gene responsible for the stage-specific 93F early puff. The E93 mRNA displays no response to ecdysteroids in late larval salivary glands but is directly induced 12 hr later by the prepupal ecdysteroid pulse, identical to the response of the 93F puff. In tissues other than the salivary gland, however, E93 displays complex spatial and temporal regulation. E93 spans at least 55 kb of genomic DNA and encodes a 146-kDa protein that has no matches in the sequence databases, but displays several characteristics of known Drosophila transcription factors. We propose that E93 acts in a stage-specific regulatory hierarchy in the salivary gland to direct its histolysis in response to the prepupal ecdysteroid pulse.

Amino Acid Sequence↗
PubMed2025

3D STED Imaging of Isolated Arabidopsis thaliana Nuclei.

Microscopy imaging of chromatin offers valuable insights into its spatial organization in the nucleus, a novel epigenetic dimension influencing the genome's functions. Particularly, visualization at the nanoscale in single cells is uniquely complementary to molecular profiling methods averaging chromatin configuration and composition over thousands of cells. How are chromatin and chromosomal domains distributed in relation to gene expression? How variable are these configurations? How do chromatin domains evolve in structure, composition, and distribution during cellular differentiation or cellular responses to environmental stimuli? Super-resolution microscopy techniques, like stimulated emission depletion (STED), are key in answering such questions. However, such imaging techniques are not often used in the field of plant cell biology compared to mammalian counterparts, which has greatly advanced our understanding of the 3D principles in genome organization. In an effort to bridge this gap, we provide a clear guide for isolating, embedding, immunostaining, and STED imaging intact leaf nuclei from Arabidopsis thaliana in 3D.

Arabidopsis↗
PubMed1995

Well-defined genome architecture in the human sperm nucleus.

Using fluorescence in situ hybridization, conventional epifluorescence microscopy, and laser scanning confocal microscopy followed by three-dimensional reconstruction we describe a well-defined higher order packaging of the human genome in the sperm cell nucleus. This was determined by the spatial localization of centromere and telomere regions of all chromosomes and supported by localization of subtelomere sequences of chromosome 3 and the entire chromosome 2. The nuclear architecture in the human sperm is characterized by the clustering of the 23 centromeres into a compact chromocenter positioned well inside the nucleus. The ends of the chromosomes are exposed to the nuclear periphery where both the subtelomere and the telomere sequences of the chromosome arms are joined into dimers. Thus chromosomes in the human sperm nucleus are looped into a hairpin-like configuration. The biological implications of this nuclear architecture in spermatogenesis and male pronuclear formation following fertilization are discussed.

Cell Nucleus↗
PubMed2004

Variability and genetic structure of the population of watermelon mosaic virus infecting melon in Spain.

The genetic structure of the population of Watermelon mosaic virus (WMV) in Spain was analysed by the biological and molecular characterisation of isolates sampled from its main host plant, melon. The population was a highly homogeneous one, built of a single pathotype, and comprising isolates closely related genetically. There was indication of temporal replacement of genotypes, but not of spatial structure of the population. Analyses of nucleotide sequences in three genomic regions, that is, in the cistrons for the P1, cylindrical inclusion (CI) and capsid (CP) proteins, showed lower similar values of nucleotide diversity for the P1 than for the CI or CP cistrons. The CI protein and the CP were under tighter evolutionary constraints than the P1 protein. Also, for the CI and CP cistrons, but not for the P1 cistron, two groups of sequences, defining two genetic strains, were apparent. Thus, different genomic regions of WMV show different evolutionary dynamics. Interestingly, for the CI and CP cistrons, sequences were clustered into two regions of the sequence space, defining the two strains above, and no intermediary sequences were identified. Recombinant isolates were found, accounting for at least 7% of the population. These recombinants presented two interesting features: (i) crossover points were detected between the analysed regions in the CI and CP cistrons, but not between those in the P1 and CI cistrons, (ii) crossover points were not observed within the analysed coding regions for the P1, CI or CP proteins. This indicates strong selection against isolates with recombinant proteins, even when originated from closely related strains. Hence, data indicate that genotypes of WMV, generated by mutation or recombination, outside of acceptable, discrete, regions in the evolutionary space, are eliminated from the virus population by negative selection.

Citrullus↗
PubMed2003

The Nuclear Factor I (NFI) gene family in mammary gland development and function.

Mammary gland development and function require the coordinated spatial and temporal expression of a large fraction of the mammalian genome. A number of site-specific transcription factors are essential to achieve the appropriate growth, branching, expansion, and involution of the mammary gland throughout early postnatal development and the lactation cycle. One family of transcription factors proposed to play a major role in the mammary gland is encoded by the Nuclear Factor I (NFI) genes. The NFI gene family is found only in multicellular animals, with single genes being present in flies and worms and four genes in vertebrates. While the NFI family expanded and diversified prior to the evolution of the mammary gland, it is clear that several mammary-gland specific genes are regulated by NFI proteins. Here we address the structure and evolution of the NFI gene family and examine the role of the NFI transcription factors in the expression of mammary-gland specific proteins, including whey acidic protein and carboxyl ester lipase. We discuss current data showing that unique NFI proteins are expressed during lactation and involution and suggest that the NFI gene family likely has multiple important functions throughout mammary gland development.

Animals↗
PubMed2007

Evolution of plant microRNA gene families.

MicroRNAs (miRNAs) are important post-transcriptional regulators of their target genes in plants and animals. miRNAs are usually 20-24 nucleotides long. Despite their unusually small sizes, the evolutionary history of miRNA gene families seems to be similar to their protein-coding counterparts. In contrast to the small but abundant miRNA families in the animal genomes, plants have fewer but larger miRNA gene families. Members of plant miRNA gene families are often highly similar, suggesting recent expansion via tandem gene duplication and segmental duplication events. Although many miRNA genes are conserved across plant species, the same gene family varies significantly in size and genomic organization in different species, which may cause dosage effects and spatial and temporal differences in target gene regulations. In this review, we summarize the current progress in understanding the evolution of plant miRNA gene families.

Animals↗
PubMed2025

Repeated evolution of photoperiodic plasticity by different genetic architectures during recurrent colonizations in a butterfly.

In cases of recurrent colonizations of similar habitats from the same base population, it is commonly expected that repeated phenotypic adaptation is caused by parallel changes in genetic variation. However, it is becoming increasingly clear that similar phenotypic variation may also evolve by alternative genetic pathways. Here, we explore the repeated evolution of photoperiodic plasticity for diapause induction across Swedish populations of the speckled wood butterfly, Pararge aegeria. This species has colonized Scandinavia at least twice, and population genomic results show that one of the candidate regions associated with spatial variation in photoperiodism is situated on the Z-chromosome. Here, we assay hybrid crosses between several populations that differ in photoperiodic plasticity for sex-linked inheritance of the photoperiodic reaction norm. We find that while a cross between more distantly related populations from the two different colonization events shows strong sex-dependent inheritance of photoperiodic plasticity, a cross between two more closely related populations within the oldest colonization range shows no such effect. We conclude that the genotype-phenotype map for photoperiodic plasticity varies across these populations and that similar local phenotypic adaptation has evolved during recurrent colonization events by partly non-parallel genetic changes.

Butterflies↗
PubMed2006

An autoregulatory loop directs the tissue-specific expression of p63 through a long-range evolutionarily conserved enhancer.

p63, a p53 family member, is essential for the development of various stratified epithelia and is one of the earliest markers of many ectodermal structures, including the epidermis, oral mucosa, apical ectodermal ridge, and mammary gland. Genetic regulatory mechanisms controlling p63 spatial expression during development have not yet been defined. Using a genomic approach, we identified an evolutionarily conserved cis-regulatory element, located 160 kb downstream of the first p63 exon, which functions as a keratinocyte-specific enhancer and is sufficient to recapitulate expression of the endogenous gene during mouse embryogenesis. Dissection of the p63 enhancer activity revealed a positive autoregulatory loop in which the p63 proteins directly bind to and are essential regulators of the enhancer. Accordingly, transactivating p63 isoforms induce endogenous p63 expression in cells that do not normally express this gene, whereas dominant negative isoforms suppress p63 expression in keratinocytes. In addition the transcription factor AP-2 also binds to the enhancer and cooperates with p63 to induce its activity. These results demonstrate that a long-range autoregulatory loop is involved in the regulation of p63 expression during embryonic development and in adult cells.

Amino Acid Sequence↗
PubMed2001

Identification, sequence and expression of a crustacean cardioactive peptide (CCAP) gene in the moth Manduca sexta.

The crustacean cardioactive peptide (CCAP) gene was isolated from the tobacco hawkmoth Manduca sexta. The gene has an open reading frame of 125 amino acid residues containing a single, complete copy of CCAP. Analysis of the gene structure revealed three introns interrupting the coding region. A comparison of the M. sexta CCAP gene with the Drosophila melanogaster genome database reveals significant similarities in sequence and gene structure. The spatial and temporal expression patterns of the CCAP gene in the M. sexta central nervous system were determined in all major post-embryonic stages using in situ hybridization techniques. The CCAP gene is expressed in a total of 116 neurons in the post-embryonic M. sexta central nervous system. Nine pairs of cells are observed in the brain, 4.5 pairs in the subesophageal ganglion, three pairs in each thoracic ganglion (T1-T3), three pairs in the first abdominal ganglion (A1), five pairs each in the second to sixth abdominal ganglia (A2-A6) and 7.5 pairs in the terminal ganglion. The CCAP gene is expressed in every ganglion in each post-embryonic stage, except in the thoracic ganglia of first- and second-instar larvae. The number of cells expressing the CCAP gene varies during post-embryonic life, starting at 52 cells in the first instar and reaching a maximum of 116 shortly after pupation. One set of thoracic neurons expressing CCAP mRNA shows unusual variability in expression levels immediately prior to larval ecdysis. Using previously published CCAP immunocytochemical data, it was determined that 91 of 95 CCAP-immunopositive neurons in the M. sexta central nervous system also express the M. sexta CCAP gene, indicating that there is likely to be only a single CCAP gene in M. sexta.

Amino Acid Sequence↗