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At least 1,081 records · Page 60Linked to original sources
PubMed2003

Large-scale characterization of genes specific to the larval nervous system in the ascidian Ciona intestinalis.

The central and peripheral nervous systems (CNS and PNS) of the ascidian tadpole larva are comparatively simple, consisting of only about 350 cells. However, studies of the expression of neural patterning genes have demonstrated overall similarity between the ascidian CNS and the vertebrate CNS, suggesting that the ascidian CNS is sufficiently complex to be relevant to those of vertebrates. Recent progress in the Ciona intestinalis genome project and cDNA project together with considerable EST information has made Ciona an ideal model for investigating molecular mechanisms underlying the formation and function of the chordate nervous system. Here, we characterized 56 genes specific to the nervous system by determining their full-length cDNA sequences and confirming their spatial expression patterns. These genes included those that function in the nervous systems of other animals, especially those involved in photoreceptor-mediated signaling and neurotransmitter release. Thus, the nervous system-specific genes in Ciona larvae will provide not only probes for determining their function but also clues for exploring the complex network of nervous system-specific genes.

Animals↗
PubMed2006

The sorting direct method for stochastic simulation of biochemical systems with varying reaction execution behavior.

A key to advancing the understanding of molecular biology in the post-genomic age is the development of accurate predictive models for genetic regulation, protein interaction, metabolism, and other biochemical processes. To facilitate model development, simulation algorithms must provide an accurate representation of the system, while performing the simulation in a reasonable amount of time. Gillespie's stochastic simulation algorithm (SSA) accurately depicts spatially homogeneous models with small populations of chemical species and properly represents noise, but it is often abandoned when modeling larger systems because of its computational complexity. In this work, we examine the performance of different versions of the SSA when applied to several biochemical models. Through our analysis, we discover that transient changes in reaction execution frequencies, which are typical of biochemical models with gene induction and repression, can dramatically affect simulator performance. To account for these shifts, we propose a new algorithm called the sorting direct method that maintains a loosely sorted order of the reactions as the simulation executes. Our measurements show that the sorting direct method performs favorably when compared to other well-known exact stochastic simulation algorithms.

Algorithms↗
PubMed2006

Identification and developmental expression of the ets gene family in the sea urchin (Strongylocentrotus purpuratus).

A systematic search in the available scaffolds of the Strongylocentrotus purpuratus genome has revealed that this sea urchin has 11 members of the ets gene family. A phylogenetic analysis of these genes showed that almost all vertebrate ets subfamilies, with the exception of one, so far found only in mammals, are each represented by one orthologous sea urchin gene. The temporal and spatial expression of the identified ETS factors was also analyzed during embryogenesis. Five ets genes (Sp-Ets1/2, Sp-Tel, Sp-Pea, Sp-Ets4, Sp-Erf) are also maternally expressed. Three genes (Sp-Elk, Sp-Elf, Sp-Erf) are ubiquitously expressed during embryogenesis, while two others (Sp-Gabp, Sp-Pu.1) are not transcribed until late larval stages. Remarkably, five of the nine sea urchin ets genes expressed during embryogenesis are exclusively (Sp-Ets1/2, Sp-Erg, Sp-Ese) or additionally (Sp-Tel, Sp-Pea) expressed in mesenchyme cells and/or their progenitors. Functional analysis of Sp-Ets1/2 has previously demonstrated an essential role of this gene in the specification of the skeletogenic mesenchyme lineage. The dynamic, and in some cases overlapping and/or unique, developmental expression pattern of the latter five genes suggests a complex, non-redundant function for ETS factors in sea urchin mesenchyme formation and differentiation.

Animals↗
PubMed1996

Kinetics of neurotensin gene expression in the developing gut.

BACKGROUND: Expression of the gene encoding neurotensin (NT/N) is regulated in a strict temporal- and spatial-specific pattern during gut development; the mechanisms (that is, transcriptional versus posttranscriptional) responsible for this expression pattern are not known. The purpose of this study was to determine whether developmental changes in NT/N expression reflect alternations in gene transcription. METHODS: Sensitive ribonuclease protection assays were performed with a rat NT/N genomic probe containing the entire sequence of both exon 1 and intron 1 hybridized with RNA from fetal (day 19) and postnatal (days 14, 28, and 60) rat jejunum and ileum; signals were quantitated densitometrically. RESULTS: Mature (exon 1) and precursor (exon 1 + intron 1) NT/N RNA, initially low in the fetus, increased dramatically by postnatal day 14 and attained maximal levels by day 28. NT/N RNA levels remained stable in the ileum of the 60-day-old rat but decreased in the jejunum, consistent with the typical expression pattern in the gut. CONCLUSIONS: Concomitant changes in expression of precursor and mature NT/N RNA suggest that NT/N gene regulation occurs at the level of transcription in the gut during development. Identifying the factors that regulate NT/N gene transcription is crucial to our understanding of how neurotensin functions in the gut.

Aging↗
PubMed2005

Regulation of poly(ADP-ribose) polymerase-1 by DNA structure-specific binding.

Poly(ADP-ribose) polymerase-1 (PARP-1) is an intracellular sensor of DNA strand breaks and plays a critical role in cellular responses to DNA damage. In normally functioning cells, PARP-1 enzymatic activity has been linked to the alterations in chromatin structure associated with gene expression. However, the molecular determinants for PARP-1 recruitment to specific sites in chromatin in the absence of DNA strand breaks remain obscure. Using gel shift and enzymatic footprinting assays and atomic force microscopy, we show that PARP-1 recognizes distortions in the DNA helical backbone and that it binds to three- and four-way junctions as well as to stably unpaired regions in double-stranded DNA. PARP-1 interactions with non-B DNA structures are functional and lead to its catalytic activation. DNA hairpins, cruciforms, and stably unpaired regions are all effective co-activators of PARP-1 auto-modification and poly(ADP-ribosyl)ation of histone H1 in the absence of free DNA ends. Enzyme kinetic analyses revealed that the structural features of non-B form DNA co-factors are important for PARP-1 catalysis activated by undamaged DNA. K0.5 constants for DNA co-factors, which are structurally different in the degree of base pairing and spatial DNA organization, follow the order: cruciform<or=hairpin<<loop. DNA structure also influenced the reaction rate; when a hairpin was substituted with a stably unpaired region, the maximum reaction velocity decreased almost 2-fold. These data suggest a link between PARP-1 binding to non-B DNA structures in genome and its function in the dynamics of local modulation of chromatin structure in the normal physiology of the cell.

Binding Sites↗
PubMed1998

Selectivity, sharing and competitive interactions in the regulation of Hoxb genes.

The clustered organisation of Hox complexes is highly conserved in vertebrates and the reasons for this are believed to be linked with the regulatory mechanisms governing their expression. In analysis of the Hoxb4-Hoxb6 region of the HoxB complex we identified enhancers which lie in the intergenic region between Hoxb4 and Hoxb5, and which are capable of mediating the correct boundaries of neural and mesodermal expression for Hoxb5. We examined their regulatory properties in the context of the local genomic region spanning the two genes by transgenic analysis, in which each promoter was independently marked with a different reporter, to monitor simultaneously the relative transcriptional read-outs from each gene. Our analysis revealed that within this intergenic region: (i) a limb and a neural enhancer selectively activate Hoxb4 as opposed to Hoxb5; (ii) a separate neural enhancer is able to activate both genes, but expression is dependent upon competition between the two promoters for the enhancer and is influenced by the local genomic context; (iii) mesodermal enhancer activities can be shared between the genes. We found similar types of regulatory interactions between Hoxb5 and Hoxb6. Together these results provide evidence for three separate general mechanisms: selectivity, competition and sharing, that control the balance of cis-regulatory interactions necessary for generating the proper spatial and temporal patterns of Hox gene expression. We suggest that these mechanisms are part of a regulatory basis for maintenance of Hox organisation.

Alkaline Phosphatase↗
PubMed1996

Dosage-sensitive maternal modifiers of the drosophila segmentation gene runt.

The protein encoded by the pair-rule gene runt functions as a transcriptional regulator during anterior-posterior patterning of the Drosophila embryo. Results of over-expression experiments as well as parallels drawn from the recent characterization of vertebrate homologues indicate that interactions with other proteins are likely to be central to the function of the Runt protein. To identify factors important for runt activity, we took advantage of an adult visible phenotype observed in animals heterozygous for runt mutations. Using a set of 126 different deficiency chromosomes we screened approximately 65% of the genome for genes that act as dose-sensitive maternal modifiers of runt. Eighteen deficiencies representing 12 putative loci were identified as maternally acting enhancers of runt haplo-insufficiency. Further characterization of two of these regions led to the identification of the interacting loci. Both of these loci affect the spatial regulation of runt transcription and appear genetically complex. Furthermore, the effects of one of these loci, M(1)1B, is indirect and mediated through effects on the transcriptional regulation of posterior gap genes.

Animals↗
PubMed1989

Spatial and temporal distribution of bovine herpesvirus 1 transcripts.

Northern (RNA) blot analysis was used to determine the spatial and temporal distribution of bovine herpesvirus 1 (BHV-1) transcripts. Total RNA was isolated from Madin-Darby bovine kidney cells which had been infected with BHV-1.2b strain K22 or BHV-1.1 strain Jura in the presence or absence of metabolic inhibitors. Cloned restriction fragments representing the entire genome of strain K22 were labeled with 32P and hybridized to immobilized RNA. A total of 54 BHV-1 transcripts were found, ranging in size from 0.4 to larger than 8 kilobases (kb). The inverted repeat regions and an adjacent segment of the unique large part of the BHV-1 genome encoded three major immediate-early (IE) transcripts and one minor IE transcript enriched after cycloheximide treatment of infected cells. Late transcripts were identified by drastically reduced abundance after cytosine arabinoside (araC) treatment. Twelve late transcripts were encoded mainly by the unique long genome region, with a cluster of four transcripts located on HindIII fragment K (map units 0.677 to 0.733). The 21 transcripts unaffected by araC treatment were defined as early; they showed dispersed locations over the whole genome, with a cluster on the unique short sequence. The 17 remaining transcripts could not be classified unambiguously as early or late by these techniques. The IE transcript with a size of 4.2 kb exhibited homology with the single IE gene of pseudorabies virus, and the IE transcript with a size of 2.9 kb was encoded in part by the genome region known to be transcriptionally active during latency.

Animals↗
PubMedDate not supplied

What is the future of diabetic wound care?

With diabetes affecting 5% to 10% of the US population, development of a more effective treatment for chronic diabetic wounds is imperative. Clinically, the current treatment in topical wound management includes debridement, topical antibiotics, and a state-of-the-art topical dressing. State-of-the-art dressings are a multi-layer system that can include a collagen cellulose substrate, neonatal foreskin fibroblasts, growth factor containing cream, and a silicone sheet covering for moisture control. Wound healing time can be up to 20 weeks. The future of diabetic wound healing lies in the development of more effective artificial "smart" matrix skin substitutes. This review article will highlight the need for novel smart matrix therapies. These smart matrices will release a multitude of growth factors, cytokines, and bioactive peptide fragments in a temporally and spatially specific, event-driven manner. This timed and focal release of cytokines, enzymes, and pharmacological agents should promote optimal tissue regeneration and repair of full-thickness wounds. Development of these kinds of therapies will require multidisciplinary translational research teams. This review article outlines how current advances in proteomics and genomics can be incorporated into a multidisciplinary translational research approach for developing novel smart matrix dressings for ulcer treatment. With the recognition that the research approach will require both time and money, the best treatment approach is the prevention of diabetic ulcers through better foot care, education, and glycemic control.

Diabetes Complications↗
PubMed2005

A pair of Sox: distinct and overlapping functions of zebrafish sox9 co-orthologs in craniofacial and pectoral fin development.

Understanding how developmental systems evolve after genome amplification is important for discerning the origins of vertebrate novelties, including neural crest, placodes, cartilage and bone. Sox9 is important for the development of these features, and zebrafish has two co-orthologs of tetrapod SOX9 stemming from an ancient genome duplication event in the lineage of ray-fin fish. We have used a genotype-driven screen to isolate a mutation deleting sox9b function, and investigated its phenotype and genetic interactions with a sox9a null mutation. Analysis of mutant phenotypes strongly supports the interpretation that ancestral gene functions partitioned spatially and temporally between Sox9 co-orthologs. Distinct subsets of the craniofacial skeleton, otic placode and pectoral appendage express each gene, and are defective in each single mutant. The double mutant phenotype is additive or synergistic. Ears are somewhat reduced in each single mutant but are mostly absent in the double mutant. Loss-of-function animals from mutations and morpholino injections, and gain-of-function animals injected with sox9a and sox9b mRNAs showed that sox9 helps regulate other early crest genes, including foxd3, sox10, snai1b and crestin, as well as the cartilage gene col2a1 and the bone gene runx2a; however, tfap2a was nearly unchanged in mutants. Chondrocytes failed to stack in sox9a mutants, failed to attain proper numbers in sox9b mutants and failed in both morphogenetic processes in double mutants. Pleiotropy can cause mutations in single copy tetrapod genes, such as Sox9, to block development early and obscure later gene functions. By contrast, subfunction partitioning between zebrafish co-orthologs of tetrapod genes, such as sox9a and sox9b, can relax pleiotropy and reveal both early and late developmental gene functions.

Animals↗
PubMed1996

Characterization of the bone sialoprotein (BSP) gene promoter.

Bone sialoprotein is a 34 kDa phosphorylated and sulphated glycoprotein that is essentially unique to mineralizing connective tissues. Recent studies on the developmental expression of BSP mRNA and the temporo-spatial appearance of the protein during bone formation in vivo and in vitro have demonstrated that BSP is expressed by differentiated osteoblasts and that it may function in the initial nucleation of hydroxyapatite crystals in de novo bone formation. To study the cell-specific regulation of BSP we have isolated genomic clones that encompass the BSP promoter regions of both the human and rat genes. These promoters are characterized by a highly conserved region (BSP Box) that extends upstream from the transcription start site to nt -370. Within this region the immediate promoter is further characterized by a unique inverted TATA box and an inverted CCAAT box, both of which are required for basal transcriptional activity. The TATA box is overlapped by a vitamin D3 response element (VDRE) which appears to mediate vitamin D suppression of BSP gene transcription by competing with the TATA-binding protein (TBP) for occupancy of the site of the pre-initiation complex formation. Mutation of the inverted TATA box into a normal TATA sequence increases transcription slightly but does not affect the functionality of the VDRE indicating that the orientation of the TATA box is not critical for these functions. Further upstream an AP-1 site, overlapped by a steroid hormone response-like sequence, mediates down-regulation of BSP transcription induced by TPA that is abrogated by a complex interaction between Jun and the glucocorticoid receptor protein induced by dexamethasone. Thus, the characterization of approximately 3 kb of the BSP promoter and approximately 2 kb of the first intron has revealed several sites of transcriptional regulation that are important in regulating BSP expression and, consequently, bone formation.

Animals↗
PubMed1992

A graphical user interface for quantitative imaging and analysis of electrophoretic gels and autoradiograms.

DNA/GUI (DNA Graphical User Interface) is an interactive software system for rapid and efficient analysis of images of the types used in genome mapping, such as autoradiograms and electrophoretic gels. Images are digitized using a commercially available charge-coupled-device (CCD) camera system and analyzed on a graphics workstation using a menu-driven user interface. DNA/GUI features automatic lane and band detection, simultaneous display of multiple images and a unique spatial-normalization algorithm. Images and their associated data are archived and easily available for later recall. Preliminary results indicate that DNA/GUI is a useful tool in the analysis and comparison of images used in a variety of applications such as genetic-linkage analysis and DNA restriction mapping. The interactive display software is based on the X Window System and is therefore readily portable to a variety of graphics workstations.

Autoradiography↗
PubMed2025

Genome-Wide Mining of lncRNAs Reveals Their Potential Regulatory Role in the Evolution of Viviparity.

Reproduction in vertebrates usually involves egg-laying (oviparity) or live-bearing (viviparity). Oviparity is the ancestral trait from which viviparity has independently evolved more than 100 times in squamate reptiles. This transition involves a series of physiological and structural changes, including the degeneration of eggshell and the evolution of a placenta and differences in the temporal and spatial expression patterns of some functional genes that drive the structural transformation. Long non-coding RNAs (lncRNAs) play important roles in the regulation of gene expression, yet it remains unclear whether they participate in gene expression shifts during the transition from oviparity to viviparity, and if so how. Therefore, we employ deep mining to identify novel lncRNAs of a closely related oviparous-viviparous pair of lizards (Phrynocephalus przewalskii and P. vlangalii). We construct cis- and trans-regulatory networks between lncRNAs and target genes using the transcriptomic data of oviduct or uteri tissues across reproductive periods. Results show that lncRNAs that regulate eggshell gland developmental genes in the oviparous lizard are lost or less expressed in the viviparous lizard. A number of lncRNAs involved in the regulation of placental development and embryo attachment in viviparous species have no orthologs&#xa0;in oviparous species, and others show little or no expression. Accordingly, lncRNAs may play important regulatory roles in the physiological and structural changes in the transition from oviparity to viviparity. These results open doors to the further elucidation of genetic regulatory networks.

Animals↗
PubMed1994

The tomato gene for the chloroplastic Cu,Zn superoxide dismutase: regulation of expression imposed in transgenic tobacco plants by a short promoter.

The chloroplastic Cu,Zn superoxide dismutase (SOD) has an important role in the defense against damage by oxygen radicals in the chloroplasts. Here, for the first time, we report on the isolation of a genomic DNA clone from tomato that contains all the coding sequence for the chloroplastic Cu,Zn SOD as well as a 442 bp DNA fragment upstream of the translational initiation site. The latter upstream sequence has a putative TATA box and a 285 bp promoter region, 5' of the apparent transcriptional initiation and a 157 bp leader region. The coding sequence is composed of 8 exons that are interspaced by 7 introns; we termed this gene SODCp;Le:1. The 442 bp fragment was cloned into a pBI101 vector, upstream of the uidA (GUS) gene, via transcriptional fusion. Agrobacterium-mediated transformation resulted in transgenic tobacco plants. The progeny (after self-pollination) of 14 transformed plants, which expressed GUS above a threshold of 1 nmol/min per mg protein, were found to fall into two distinct groups. In the seedlings of 10 lines (group A) GUS expression was enhanced by exposure to light. In 4 lines of this group maintenance for 3 days in the dark eliminated GUS activity. The seedlings of group B expressed GUS regardless of the light/dark regime. In plants of group A, GUS expression was also developmentally regulated: high GUS activity in young leaves, low activity in mature leaves and no activity in the roots. The results suggest that this short chloroplastic Cu,Zn SOD promoter contains motifs for developmental (spatial) regulation as well as motifs responsive to light (or to oxygen radicals resulting from light-driven photosynthesis).

Amino Acid Sequence↗
PubMed2026

Low-Cost Nucleic-Acid-Based Radial Flow Assay for the Detection of GSTP1 Promoter DNA Methylation in Prostate Cancer.

DNA methylation of the glutathione S-transferase pi 1 (GSTP1) promoter is a widely studied epigenetic biomarker for prostate cancer; however, its direct detection in genomic DNA remains analytically challenging without complex chemical or amplification-based workflows. Here, we report a nucleic acid-based radial flow assay (NABRFA) that enables visual and pattern-based detection of gene-specific DNA methylation using gold nanoparticle (AuNP)-conjugated oligonucleotide probes. Thiol-modified single-stranded DNA probes targeting the GSTP1 CpG island (5'ThG) were conjugated to AuNPs to form stable probe-nanoparticle constructs that retain colloidal stability under high ionic strength conditions (0.5 M NaCl). Upon hybridization with methylation-protected GSTP1 DNA, the resulting AuNP-DNA complexes exhibit hybridization-dependent modulation of transport and retention on a porous nylon membrane, generating characteristic concentric radial patterns. These patterns arise from spatial separation between retained hybridized complexes and outwardly transported unbound probe-functionalized nanoparticles, enabling direct visual discrimination of target presence. The assay demonstrated concentration-dependent pattern evolution, with visual detection achievable down to 1 ng of target DNA and an analytically determined limit of detection of approximately 32 ng, based on image-derived gray value analysis. The human prostate cancer cell line LNCaP, known for GSTP1 promoter hypermethylation, was used as the test model for assay validation. Comparative analysis using methyl-sensitive restriction enzyme-treated native genomic DNA from the human osteosarcoma MG-63 cell line (non-prostate cancer, GSTP1 methylation-negative control) and the human lung fibroblast WI-38 cell line (non-cancerous, GSTP1 methylation-negative control) confirmed assay specificity. By coupling sequence-specific hybridization with transport-mediated nanoparticle pattern formation, NABRFA provides a label-free and conversion-free analytical strategy for detection of methylation-protected GSTP1 DNA using minimal instrumentation. This work establishes a proof-of-concept membrane-based, transport-driven sensing approach for epigenetic biomarker detection and highlights its potential for integration into simplified molecular diagnostic workflows.

Humans↗
PubMed2005

Phylogenetic analysis reveals a novel protein family closely related to adenosine deaminase.

Adenosine deaminase (ADA) is a well-characterized enzyme involved in the depletion of adenosine levels. A group of proteins with similarity to ADA, the adenosine deaminase-related growth factors (ADGF; known as CECR1 in vertebrates), has been described recently in various organisms. We have determined the phylogenetic relationships of various gene products with significant amino acid similarity to ADA using parsimony and Bayesian methods, and discovered a novel paralogue, termed ADA-like (ADAL). The ADGF proteins share a novel amino acid motif, "MPKG," within which the proline and lysine residues are also conserved in the ADAL and ADA subfamilies. The significance of this new domain is unknown, but it is located just upstream of two ADA catalytic residues, of which all eight are conserved among the ADGF and ADAL proteins. This conservation suggests that ADGF and ADAL may share the same catalytic function as ADA, which has been proven for some ADGF members. These analyses also revealed that some genes previously thought to be classic ADAs are instead ADAL or ADGFs. We here define the ADGF, ADAL, ADA, adenine deaminase (ADE), and AMP deaminase (AMPD) groups as subfamilies of the adenyl-deaminase family. The availability of genomic data for the members of this family allowed us to reconstruct the intron evolution within the phylogeny and strengthen the introns-late hypothesis of the synthetic introns theory. This study shows that ADA activity is clearly more complex than once thought, perhaps involving a delicately balanced pattern of temporal and spatial expression of a number of paralogous proteins.

Adenosine Deaminase↗
PubMed1984

Drosophila melanogaster U1 snRNA genes.

We have isolated and characterized a recombinant which contains a Drosophila melanogaster U1 small nuclear RNA (snRNA) gene colinear with the published snRNA sequence. Southern hybridizations of the fly genomic DNA, using as probe a plasmid containing only the coding region of the gene, shows that the fly contains at most three or four genes and very few related sequences for the small nuclear U1 RNA. These genes were localized by in situ hybridization at different chromosomal loci and show no spatial relationship to the U2 snRNA genes.

Animals↗
PubMed1999

Ten years of gene targeting: targeted mouse mutants, from vector design to phenotype analysis.

Gene targeting, defined as the introduction of site-specific modifications into the genome by homologous recombination, has revolutionarized the field of mouse genetics and allowed the analysis of diverse aspects of gene function in vivo. It is now possible to engineer specific genetic alterations ranging from subtle mutations to chromosomal rearrangements and more recently, even tissue-specific inducible gene targeting with temporo-spatial control has become feasible. This review tries to recapitulate what we have learned in this extremely rapidly expanding field during the past decade. Diverse aspects of the technique will be discussed starting from basic construct design to the analysis of complex phenotypes, including recent advances on inducible expression system. Many examples from different areas of biomedical research are given to illustrate the purpose and limitations of the employed experimental approaches.

Animals↗