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PubMed1993

Characterization of a gene that is expressed early in somatic embryogenesis of Daucus carota.

The EMB-1 mRNA of carrot (Daucus carota) was isolated as an embryo abundant cDNA clone (T.H. Ulrich, E.S. Wurtele, B.J. Nikolau [1990] Nucleic Acids Res 18: 2826). Northern analyses of RNA isolated from embryos, cultured cells, and a variety of vegetative organs indicate that the EMB-1 mRNA specifically accumulates in embryos, beginning at the early stages of embryo development. In situ hybridization with both zygotic and somatic embryos show that the EMB-1 mRNA begins to accumulate at low levels throughout globular embryos. Accumulation of EMB-1 mRNA increases and becomes more localized as embryos mature; in torpedo embryos, EMB-1 mRNA preferentially accumulates in the meristematic regions, particularly the procambium. The similarity in distribution of EMB-1 mRNA in both zygotic and somatic embryos indicates that much of the spatial pattern of expression of the emb-1 gene is dependent on the developmental program of the carrot embryo and does not require maternal or endosperm factors. The EMB-1 protein (relative molecular weight 9910) is a very hydrophilic protein that is a member of a class of highly conserved proteins (typified also by the Em protein of wheat and the Lea D19 protein of cotton) that may be ubiquitous among angiosperm embryos but whose functions are as yet unknown. The carrot genome appears to contain one or two copies of the emb-1 gene. A 1313-base pair DNA fragment of the carrot genome containing the emb-1 gene was isolated and sequenced. The gene is interrupted by a single intron of 99 base pairs. Primer extension experiments identify two EMB-1 mRNAs, differing by 6 bases at their 5' ends that are transcribed from this gene.

Amino Acid Sequence↗

PubMed2024

DNA methylation reprogramming in teleosts.

Early embryonic development is crucially important but also remarkably diverse among animal taxa. Axis formation and cell lineage specification occur due to both spatial and temporal control of gene expression. This complex system involves various signaling pathways and developmental genes such as transcription factors as well as other molecular interactants that maintain cellular states, including several types of epigenetic marks. 5mC DNA methylation, the chemical modification of cytosines in eukaryotes, represents one such mark. By influencing the compaction of chromatin (a high-order DNA structure), DNA methylation can either repress or induce transcriptional activity. Mammals exhibit a reprogramming of DNA methylation from the parental genomes in the zygote following fertilization, and later in primordial germ cells (PGCs). Whether these periods of methylation reprogramming are evolutionarily conserved, or an innovation in mammals, is an emerging question. Looking into these processes in other vertebrate lineages is thus important, and teleost fish, with their extensive species richness, phenotypic diversity, and multiple rounds of whole genome duplication, provide the perfect research playground for answering such a question. This review aims to present a concise state of the art of DNA methylation reprogramming in early development in fish by summarizing findings from different research groups investigating methylation reprogramming patterns in teleosts, while keeping in mind the ramifications of the methodology used, then comparing those patterns to reprogramming patterns in mammals.

Animals↗

PubMed2000

A new mutant class, made by targeted mutagenesis, of phage PRD1 reveals that protein P5 connects the receptor binding protein to the vertex.

Phage PRD1 and adenovirus share a number of structural and functional similarities, one of which is the vertex organization at the fivefold-symmetry positions. We developed an in vitro mutagenesis system for the linear PRD1 genome in order to make targeted mutations. The role of protein P5 in the vertex structure was examined by this method. Mutation in gene V revealed that protein P5 is essential. The absence of P5 did not compromise the particle assembly or DNA packaging but led to a deficient vertex structure where the receptor binding protein P2, in addition to protein P5, was missing. P5(-) particles also lost their DNA upon purification. Based on this and previously published information we propose a spatial model for the spike structure at the vertices. This resembles to the corresponding structure in adenovirus.

Adsorption↗

PubMed2005

Hormone-driven mechanisms in the central nervous system facilitate the analysis of mammalian behaviours.

Hormonal effects on behaviours in animals and humans are now well enough understood for general statements about causal steps to be proposed. Facilitation or repression of a given behaviour by a given hormone can depend on the person's genetic and developmental history, on the temporal and spatial parameters of the hormone's administration, on the hormone's metabolism and on the specific receptor isoform available in a given neuron. The gene for oestrogen receptor-alpha is required for an entire chain of behaviours essential for reproduction, from courtship through maternal behaviours. In order to show that it is possible to use endocrine tools to explain a mammalian behaviour, we analysed lordosis behaviour neuronal circuitry as well as the molecular mechanisms of its facilitation by oestrogens. The functional genomics of oestrogenic effects on lordosis arrange themselves in modules for neuronal Growth, Amplification (by progestins), Preparatory behaviours, Permissive actions by hypothalamic neurons, and Synchronization of mating behaviour with ovulation (GAPPS). A related four-gene micronet involving the amygdala and the paraventricular nucleus of the hypothalamus supports social recognition. Underlying all sociosexual behaviour is the fundamental arousal of brain and behaviour. Elementary arousal depends on a bilateral, bidirectional system universal among mammalian brains, and it can be altered by null deletion of the gene for oestrogen receptor-alpha. Future molecular and biophysical studies will specify how hormone effects in the brain change central nervous system state in such a manner as to alter the frequencies of entire sets of behavioural responses.

Animals↗

PubMed2006

The fitness effects of outcrossing in Calylophus serrulatus, a permanent translocation heterozygote.

Small and relatively isolated populations that occupy fragmented habitat are at risk of local extinction. However, fitness consequences of fragmentation related to mating distance, such as inbreeding depression following increased self- and near-neighbor mating, may not follow standard expectations in species with specialized genetic systems. We investigated the effect of mating distance on progeny fitness in Calylophus serrulatus, a primarily autogamous, permanent translocation heterozygote that is restricted to prairie fragments in the North American tallgrass prairie region. We pollinated flowers by hand in the field with pollen sampled at various distances from the maternal parent within and between three populations in southeastern Minnesota. We raised the progeny in a greenhouse and measured fitness-related characters. Because their genetic system prevents loss of heterozygosity throughout much of the genome, regardless of inbreeding, permanent translocation heterozygotes are not expected to exhibit inbreeding depression. Consistent with this expectation, in no case did progeny of self matings suffer significantly reduced mean fitness compared to progeny from crosses between plants. Crosses between plants in the two closely situated (2 km) populations yielded progeny with fitness intermediate to their parents, but crosses between each of those populations and the more distant (20 km) population yielded progeny with reduced fitness, suggesting outbreeding depression at this largest spatial scale. Similarly, fitness of self-pollinated progeny and progeny from "near" crosses (<2 m) within populations tended to be higher than "mid" (10-25 m) and "far" (>35 m) cross-progeny fitness. Under the current conditions of fragmentation, it seems likely that the distant matings that produce outbreeding depression are rare. It appears that mean fitness in this species is maintained in the context of severe fragmentation of its populations, largely because of its genetic system.

Crosses, Genetic↗

PubMed1996

Poisson process approximation for sequence repeats, and sequencing by hybridization.

Sequencing by hybridization is a tool to determine a DNA sequence from the unordered list of all l-tuples contained in this sequence; typical numbers for l are l = 8, 10, 12. For theoretical purposes we assume that the multiset of all l-tuples is known. This multiset determines the DNA sequence uniquely if none of the so-called Ukkonen transformations are possible. These transformations require repeats of (l-1)-tuples in the sequence, with these repeats occurring in certain spatial patterns. We model DNA as an i.i.d. sequence. We first prove Poisson process approximations for the process of indicators of all leftmost long repeats allowing self-overlap and for the process of indicators of all left-most long repeats without self-overlap. Using the Chen-Stein method, we get bounds on the error of these approximations. As a corollary, we approximate the distribution of longest repeats. In the second step we analyze the spatial patterns of the repeats. Finally we combine these two steps to prove an approximation for the probability that a random sequence is uniquely recoverable from its list of l-tuples. For all our results we give some numerical examples including error bounds.

Algorithms↗

PubMed2003

Whole-genome microarrays of fission yeast: characteristics, accuracy, reproducibility, and processing of array data.

BACKGROUND: The genome of the fission yeast Schizosaccharomyces pombe has recently been sequenced, setting the stage for the post-genomic era of this increasingly popular model organism. We have built fission yeast microarrays, optimised protocols to improve array performance, and carried out experiments to assess various characteristics of microarrays. RESULTS: We designed PCR primers to amplify specific probes (180-500 bp) for all known and predicted fission yeast genes, which are printed in duplicate onto separate regions of glass slides together with control elements (approximately 13,000 spots/slide). Fluorescence signal intensities depended on the size and intragenic position of the array elements, whereas the signal ratios were largely independent of element properties. Only the coding strand is covalently linked to the slides, and our array elements can discriminate transcriptional direction. The microarrays can distinguish sequences with up to 70% identity, above which cross-hybridisation contributes to the signal intensity. We tested the accuracy of signal ratios and measured the reproducibility of array data caused by biological and technical factors. Because the technical variability is lower, it is best to use samples prepared from independent biological experiments to obtain repeated measurements with swapping of fluorochromes to prevent dye bias. We also developed a script that discards unreliable data and performs a normalization to correct spatial artefacts. CONCLUSIONS: This paper provides data for several microarray properties that are rarely measured. The results define critical parameters for microarray design and experiments and provide a framework to optimise and interpret array data. Our arrays give reproducible and accurate expression ratios with high sensitivity. The scripts for primer design and initial data processing as well as primer sequences and detailed protocols are available from our website.

DNA Primers↗

PubMed2000

Cloning and characterization of zebrafish smad2, smad3 and smad4.

smad genes encode transcription factors involved in the signal transduction of members of the TGFbeta superfamily. We report here the cloning, characterization and genomic mapping of smad2, smad3 and smad4 from the zebrafish, Danio rerio. In Xenopus, smad2 overexpression has been shown to interfere with gastrulation and dorsal cell fate specification. However, full-length zebrafish smad2, although functionally active in Xenopus explants, has no effect when overexpressed in zebrafish embryos. In contrast, an N-terminally truncated, constitutively active version of Smad2 protein causes severe dorsalization or partial secondary axis formation, pointing to a role of Smad2 during mesoderm and axis formation. The temporal and spatial expression patterns of zebrafish smad2, 3 and 4 were investigated by developmental RT-PCR and whole mount in-situ hybridization. All three genes show strong and ubiquitous maternal expression. Zygotic expression is weak and ubiquitous in the case of smad2, and strong and ubiquitious in the case of smad4, while smad3 shows a spatially restricted zygotic expression pattern. It is expressed in migrating neural crest cells of the trunk and a subset of cells in the diencephalon in close proximity to the expression domain of the Nodal-related protein Cyclops/Ndr2/Znr1, a potential signal upstream of Smad2/3 required for eye-field separation and floor plate specification. Overexpression of truncated smad2 in cyclops mutant embryos leads to a rescue of the eye and floorplate defects. These data suggest that Smad2 acts as a mediator of Nodal signals during zebrafish midline signaling, while Smad3 might be involved in later steps of eye field separation.

Amino Acid Sequence↗

PubMed1987

Putative polyadenylation signals in nuclear genes of higher plants: a compilation and analysis.

In animal and viral pre-mRNAS, the process of polyadenylation is mediated through several cis-acting poly (A) signals present upstream and downstream from poly (A) sites. The situation regarding polyadenylation of higher plant pre-mRNAS, however, has remained obscure so far. In this paper, a search for putative poly (A) signals is made by considering the published data from 46 plant genomic DNA sequences. Certain domains in the 3' untranslated regions from nuclear genes of higher plants were compiled and occurrence of sequence motifs such as AATAAA, CAYTG, YGTGTTYY and YAYTG was scored in relation to poly (A) sites. Moreover, consensus sequences for important regions in the 3' untranslated sequences and poly (A) signals were also deduced from the data. It was inferred that sequence motifs similar to poly (A) signals exist around poly (A) sites but some of them are in entirely different spatial relationship than observed in other eukaryotes. This indicates their probable non-involvement in the process of polyadenylation in higher plants necessitating a functional analysis approach to define the plant specific poly (A) signals.

Animals↗

PubMedDate not supplied

Chromosome variability and germ cell development in the house mouse.

Structural heterozygosities of the karyotype have detrimental effects on the meiotic process, resulting very often in impairment of fertility in the carriers. Both male and female germ cell development are affected by chromosomal variability although spermatogenesis seems particularly prone to be affected, probably because of the intrinsic characteristics of the male germ cell cytodifferentiation process (i.e. the histological architecture of the seminiferous epithelium). However, euploid and aneuploid sperm do not seem to differ in the molecular organization of the genome they carry, thus explaining the almost regular capacity to accomplish the first zygotic developmental stages by the aneuploid sperm (aneuploid both for gametogenic genes and for entire chromosomal arms). A survey of the molecular and morphological data available on germ cell development in conditions of chromosomal rearrangement leads to the conclusion that the current hypotheses accounting for this phenomenon can only partly explain it. A working hypothesis is proposed which considers the three-dimensional changes (produced by structural heterozygosity) in the spatial order of chromosomes within the nucleus as the primary cause potentially able to trigger distorted functioning of the germ cells.

Aneuploidy↗

PubMed2003

Cell death in Pseudomonas aeruginosa biofilm development.

Bacteria growing in biofilms often develop multicellular, three-dimensional structures known as microcolonies. Complex differentiation within biofilms of Pseudomonas aeruginosa occurs, leading to the creation of voids inside microcolonies and to the dispersal of cells from within these voids. However, key developmental processes regulating these events are poorly understood. A normal component of multicellular development is cell death. Here we report that a repeatable pattern of cell death and lysis occurs in biofilms of P. aeruginosa during the normal course of development. Cell death occurred with temporal and spatial organization within biofilms, inside microcolonies, when the biofilms were allowed to develop in continuous-culture flow cells. A subpopulation of viable cells was always observed in these regions. During the onset of biofilm killing and during biofilm development thereafter, a bacteriophage capable of superinfecting and lysing the P. aeruginosa parent strain was detected in the fluid effluent from the biofilm. The bacteriophage implicated in biofilm killing was closely related to the filamentous phage Pf1 and existed as a prophage within the genome of P. aeruginosa. We propose that prophage-mediated cell death is an important mechanism of differentiation inside microcolonies that facilitates dispersal of a subpopulation of surviving cells.

Bacterial Proteins↗

PubMed2004

Genetically engineered mouse models for lung cancer.

The lung is a complex organ consisting of numerous cell types that function to ensure sufficient gas exchange to oxygenate the blood. In order to accomplish this function, the lung must be exposed to the external environment and at the same time maintain a homeostatic balance between its function in gas exchange and the maintenance of inflammatory balance. During the past two decades, as molecular methodologies have evolved with the sequencing of entire genomes, the use of in vivo models to elucidate the molecular mechanisms involved in pulmonary physiology and disease have increased. The mouse has emerged as a potent model to investigate pulmonary physiology due to the explosion in molecular methods that now allow for the developmental and tissue-specific regulation of gene transcription. Initial efforts to manipulate gene expression in the mouse genome resulted in the generation of transgenic mice characterized by the constitutive expression of a specific gene and knockout mice characterized by the ablation of a specific gene. The utility of these original mouse models was limited, in many cases, by phenotypes resulting in embryonic or neonatal lethality that prevented analysis of the impact of the genetic manipulation on pulmonary biology. Second-generation transgenic mouse models employ multiple strategies that can either activate or silence gene expression thereby providing extensive temporal and spatial control of the experimental parameters of gene expression. These highly regulated mouse models are intended to serve as a foundation for further investigation of the molecular basis of human disease such as tumorigenesis. This review describes the principles, progress, and application of systems that are currently employed in the conditional regulation of gene expression in the investigation of lung cancer.

Animals↗

PubMed2026

Efferocytosis related KCTD12 is a clinico-immune target in lung adenocarcinoma.

BACKGROUND: Efferocytosis, the clearance of apoptotic cells by phagocytes, contributes to immune homeostasis but may also promote tumor immune tolerance. However, its transcriptional landscape and clinical relevance in lung adenocarcinoma (LUAD) remain incompletely understood. METHODS: We systematically analyzed efferocytosis-associated genes across TCGA and multiple GEO datasets to classify LUAD subtypes and construct a prognostic risk model. The prognostic and immunological relevance of this model was validated in four independent cohorts and further assessed through immune infiltration, genomic, and immunotherapy datasets. Functional and pharmacogenomic analyses were performed to identify potential therapeutic vulnerabilities, and the efferocytosis-associated signature gene KCTD12 was subsequently validated in vitro. RESULTS: Unsupervised clustering identified two efferocytosis-based LUAD subtypes with distinct prognostic and immune-metabolic characteristics. The derived risk model robustly predicted overall survival across validation cohorts. Among the model genes, KCTD12 emerged as an efferocytosis-associated candidate linked to an immune-active tumor microenvironment. Across the analyzed single-cell, spatial transcriptomic, and immunotherapy-treated cohorts, higher KCTD12 expression was associated with enhanced cytotoxic T-cell activity and more favorable treatment outcomes. Functional experiments confirmed that KCTD12 suppresses tumor cell proliferation, reduces colony formation, and enhances OT-1 CD8+ T-cell activation and cytotoxicity. CONCLUSIONS: Our study identifies an efferocytosis-associated transcriptional program linked to immune heterogeneity and prognosis in LUAD. The efferocytosis-related risk signature provides a framework for prognostic and immune stratification, while KCTD12 represents a candidate biomarker associated with immune activation and clinical outcomes in immunotherapy-treated cohorts. Its treatment-specific predictive value requires prospective validation in appropriately controlled studies.

KCTD12↗

PubMedDate not supplied

[Phylogeographic approach in the interpretation of mitochondrial DNA sequencing results in forensics].

In recent years, forensic mitochondrial DNA analysis has been undertaken from an evolutionary perspective. In particular, the phylogeographic approach based on a phylogenetic analysis of the spatial distribution of mitochondrial haplotypes and haplogroups appears to be a useful tool in the interpretation of identification cases. In this study, the phylogeographic approach has been employed in the analysis of three difficult forensic cases, where single nucleotide, homoplasmic differences were found between the reference and evidentiary haplotypes. mtDNA sequence variation has been examined by the control region (HVS I and HVS II) direct sequencing. Additionally, in order to clarify the subhaplogroup status of the selected haplotypes, DNA sequences of entire mitochondrial genomes obtained from two samples representing J1b subclade have been analyzed.

DNA Fingerprinting↗

PubMed1995

A rapid method for mRNA detection in single-cell biopsies from preimplantation-stage bovine embryos.

Major questions concerning the control of development and gene expression at the cellular level are still unanswered. Nowhere is this more evident than during the earliest stages of development and embryogenesis. This study describes the detection of specific gene transcripts in single cells derived from bovine embryos. Following in vitro fertilization (IVF) and in vitro culture (IVC) of bovine embryos, small groups of cells and even single blastomeres from 32 to 64-cell embryos were micromanipulated into individual tubes for analysis of cytoplasmic RNAs. Reverse transcriptase-PCR was applied to cell lysates for the amplification of beta-actin mRNA transcripts. Primers were designed to flank an intron expected to be present within genomic DNA sequences, thus allowing for simple differentiation between DNA- and RNA-derived amplification products. Using a 50-cycle amplification profile, a 260 bp band could be seen as a PCR product derived from a single blastomere following electrophoresis in an ethidium bromide-stained agarose gel. The identity of the band was verified by DNA sequence determination and diagnostic restriction digestion. Lysates derived from single blastomeres in this way have been used for simultaneously phenotyping multiple RNA products. This capability allows the spatial analysis of gene expression and development within embryos from the earliest stages of cellular differentiation.

Journal Article↗

PubMed2005

An integrative model of mouse cardiac electrophysiology from cell to torso.

AIMS: Although the transgenic mouse has become an important new tool in the study of human diseases and the design of new therapies, a complete picture of cardiac electrophysiology in the mouse, from genome to body surface, is lacking. A computational model of the mouse heart is presented, which is used to study the impact of ion-channel and structural manipulations on the distributions of extracellular potentials on the heart and body surface. METHODS: A model of the mouse heart anatomy, fibre organization and torso geometry was constructed from DTMRI images. An anisotropic bidomain model, with a modified Pandit et al. model for the ionic currents, was used to represent the electrical properties of the tissue. Spatial heterogeneity in the ion currents was introduced by modulating the transient outward current. A sinus beat was simulated in hearts with different tissue and membrane properties and the extracellular potentials were computed at both the heart and body surface. RESULTS: The simulated transmembrane patterns in the heart, and the timing and morphology of the simulated ECG waveforms were consistent with experimental measurements. In addition, the patterns of activation and recovery and the waveforms of the corresponding ECG were found to be relatively insensitive to changes in cell type distribution and tissue anisotropy. CONCLUSION: Because of the small size of the heart, an integrative model of mouse electrophysiology can be simulated from cell to torso, enabling a new tool to study how extracellular signals might be used to detect molecular changes underlying an arrhythmogenic substrate.

Algorithms↗

PubMed2000

Xylem-specific expression of wound-inducible rice peroxidase genes in transgenic plants.

A peroxidase gene, poxA, was isolated from a rice (Oryza sativa L.) genomic library. The gene consists of four exons whose combined sequences were identical to that of the prxRPA mRNA whose levels were dramatically stimulated by wounding as well as by treatment of rice shoots with ethephon or UV irradiation [H. Ito, F. Kimizuka, A. Ohbayashi, H. Matsui, M. Honma, A. Shinmyo, Y. Ohashi, A.B. Caplan, R.L. Rodriguez, Molecular cloning and characterization of two complementary DNAs encoding putative peroxidases from rice (Oryza sativa L.) shoots, Plant Cell Rep. 13 (1994) 361-366]. The temporal and spatial expression properties of the poxA gene promoter as well as that from a second related peroxidase gene, poxN, were analyzed in transgenic tobacco and rice plants using the uidA gene as a reporter. In transgenic tobacco, UV- and wound-responsive cis-elements were located within 144 bp from the translational start codon of the poxA gene. The poxN promoter, however, was inactive in the heterologous host as no significant GUS activity was evident. On the other hand, chimeric uidA genes containing 2.2 kb of the poxA promoter or 1.4 kb of poxN promoter were active in transgenic rice plants. Both peroxidase promoters directed GUS activities in a spatial and tissue specific manner coincident with the expression patterns exhibited by their mRNAs. Histochemical analysis of transgenic rice plants showed that both peroxidase genes are expressed in the vascular bundles of the shoot apex and lamina joint, and in xylem-parenchyma cells of the leaf blade and sheath.

Journal Article↗

PubMed1989

Caenorhabditis elegans has scores of homoeobox-containing genes.

Homoeobox-containing genes control cell identities in particular spatial domains, cell lineages, or cell types during the development of Drosophila and Caenorhabditis elegans, and they probably control similar processes in vertebrates. More than 80 genes with homoeoboxes that have sequence similarities ranging from 25 to 100% have been isolated by genetic means or by DNA hybridization to previously isolated genes. We synthesized 500-2,000-fold degenerate oligonucleotides corresponding to a set of well-conserved eight amino acid sequences from the helix-3 region of the homoeodomain. We screened C. elegans genomic libraries with these probes and identified 49 putative homoeobox-containing loci. DNA sequencing confirmed that eight out of ten selected loci had sequences corresponding to the conserved helix-3 region plus additional flanking sequence similarity. One of these genes contained a sequence corresponding to a complete pou-domain and another was closely related to the homoeobox-containing genes caudal/cdx-1. The putative homoeobox loci were mapped to the physical contig map of C. elegans, allowing the identification of potentially corresponding genes from the correlated genetic map. We estimate that the number of homoeobox-containing genes in C. elegans is at least 60, constituting approximately 1% of the estimated total number of genes.

Amino Acid Sequence↗