Specification of the eye field (EF) within the neural plate marks the earliest detectable stage of eye development. Experimental evidence, primarily from non-mammalian model systems, indicates that the stable formation of this group of cells requires the activation of a set of key transcription factors. This crucial event is challenging to probe in mammals and, quantitatively, little is known regarding the regulation of the transition of cells to this ocular fate.
View Article and Find Full Text PDFTopologically associating domains (TADs) have been proposed to both guide and constrain enhancer activity. is located within a TAD known to contain all its enhancers. To investigate the importance of chromatin conformation and TAD integrity on developmental gene regulation, we have manipulated the TAD - creating internal deletions, deleting CTCF sites, and deleting and inverting sequences at TAD boundaries.
View Article and Find Full Text PDFThe rate of RNA polymerase II (RNAPII) elongation has an important role in the control of alternative splicing (AS); however, the consequences of an altered elongation rate are unknown. Here, we generated mouse embryonic stem cells (ESCs) knocked in for a slow elongating form of RNAPII We show that a reduced transcriptional elongation rate results in early embryonic lethality in mice. Focusing on neuronal differentiation as a model, we observed that slow elongation impairs development of the neural lineage from ESCs, which is accompanied by changes in AS and in gene expression along this pathway.
View Article and Find Full Text PDFDNA methylation and Polycomb are key factors in the establishment of vertebrate cellular identity and fate. Here we report de novo missense mutations in DNMT3A, which encodes the DNA methyltransferase DNMT3A. These mutations cause microcephalic dwarfism, a hypocellular disorder of extreme global growth failure.
View Article and Find Full Text PDFAicardi-Goutières syndrome (AGS) provides a monogenic model of nucleic acid-mediated inflammation relevant to the pathogenesis of systemic autoimmunity. Mutations that impair ribonuclease (RNase) H2 enzyme function are the most frequent cause of this autoinflammatory disorder of childhood and are also associated with systemic lupus erythematosus. Reduced processing of eitherRNA:DNAhybrid or genome-embedded ribonucleotide substrates is thought to lead to activation of a yet undefined nucleic acid-sensing pathway.
View Article and Find Full Text PDFHuman β-defensin 3 (hBD3) is a cationic host defence peptide and is part of the innate immune response. HBD3 is present on a highly copy number variable block of six β-defensin genes, and increased copy number is associated with the autoimmune disease psoriasis. It is not known how this increase influences disease development, but psoriasis is a T cell-mediated disease and activation of the innate immune system is required for the initial trigger that leads to the amplification stage.
View Article and Find Full Text PDFConservation within intergenic DNA often highlights regulatory elements that control gene expression from a long range. How conservation within a single element relates to regulatory information and how internal composition relates to function is unknown. Here, we examine the structural features of the highly conserved ZRS (also called MFCS1) cis-regulator responsible for the spatiotemporal control of Shh in the limb bud.
View Article and Find Full Text PDFβ-defensin peptides are a family of antimicrobial peptides present at mucosal surfaces, with the main site of expression under normal conditions in the male reproductive tract. Although they kill microbes in vitro and interact with immune cells, the precise role of these genes in vivo remains uncertain. We show here that homozygous deletion of a cluster of nine β-defensin genes (DefbΔ9) in the mouse results in male sterility.
View Article and Find Full Text PDFThe presence of ribonucleotides in genomic DNA is undesirable given their increased susceptibility to hydrolysis. Ribonuclease (RNase) H enzymes that recognize and process such embedded ribonucleotides are present in all domains of life. However, in unicellular organisms such as budding yeast, they are not required for viability or even efficient cellular proliferation, while in humans, RNase H2 hypomorphic mutations cause the neuroinflammatory disorder Aicardi-Goutières syndrome.
View Article and Find Full Text PDFAntimicrob Agents Chemother
May 2010
Beta-defensins are known to be both antimicrobial and able to chemoattract various immune cells. Although the sequences of paralogous genes are not highly conserved, the core defensin structure is retained. Defb14-1C(V) has bactericidal activity similar to that of its parent peptide (murine beta-defensin Defb14) despite all but one of the canonical six cysteines being replaced with alanines.
View Article and Find Full Text PDFBeta-defensins are both antimicrobial and able to chemoattract various immune cells including immature dendritic cells and CD4 T cells through CCR6. They are short, cationic peptides with a highly conserved six-cysteine motif. It has been shown that only the fifth cysteine is critical for chemoattraction of cells expressing CCR6.
View Article and Find Full Text PDFHuman beta-defensin 2 (HBD2) has been shown to interact with pathogenic bacteria and components of the mammalian innate and adaptive immune response. We describe a quick and reliable method for the production of HBD2 in Escherichia coli. HBD2 was expressed as an insoluble fusion, chemically cleaved and oxidised to give a single, folded HBD2 beta-isoform.
View Article and Find Full Text PDFBeta-defensins comprise a family of cationic, antimicrobial and chemoattractant peptides. The six cysteine canonical motif is retained throughout evolution and the disulphide connectivities stabilise the conserved monomer structure. A murine beta-defensin gene (Defr1) present in the main defensin cluster of C57B1/6 mice, encodes a peptide with only five of the canonical six cysteine residues.
View Article and Find Full Text PDFBackground: We have examined the evolution of the genes at the major human beta-defensin locus and the orthologous loci in a range of other primates and mouse. For the first time these data allow us to examine selective episodes in the more recent evolutionary history of this locus as well as the ancient past. We have used a combination of maximum likelihood based tests and a maximum parsimony based sliding window approach to give a detailed view of the varying modes of selection operating at this locus.
View Article and Find Full Text PDFWe describe an air-liquid interface primary culture method for murine tracheal epithelial cells on semi-permeable membranes, forming polarized epithelia with a high transepithelial resistance, differentiation to ciliated and secretory cells, and physiologically appropriate expression of key genes and ion channels. We also describe the isolation of primary murine nasal epithelial cells for patch-clamp analysis, generating polarised cells with physiologically appropriate distribution and ion channel expression. These methods enable more physiologically relevant analysis of murine airway epithelial cells in vitro and ex vivo, better utilisation of transgenic mouse models of human pulmonary diseases, and have been approved by the European Working Group on CFTR expression.
View Article and Find Full Text PDFBeta-defensins are two exon genes which encode broad spectrum antimicrobial cationic peptides. We have analyzed the largest murine cluster of these genes which localizes to chromosome 8. Using hidden Markov models, we identified six beta-defensin exon 2-like sequences and subsequently found full-length expressed transcripts for these novel genes.
View Article and Find Full Text PDFBeta-defensins comprise a family of cationic peptides, which are predominately expressed at epithelial surfaces and have a broad-range antimicrobial activity. We have assembled two BAC-based contigs from the chromosomal region 8A4 that contain the murine defensins, and we have mapped six reported beta-defensin genes. In addition, we have isolated and functionally characterized a novel beta-defensin gene that deviates from the canonical six cysteine motif present in the mature functional peptide of all other beta-defensins.
View Article and Find Full Text PDFBeta defensins are small cationic antimicrobial peptides present in the respiratory system which have been proposed to be dysfunctional in the environment of the cystic fibrosis lung. Defb1, a murine homologue to the human beta defensins, has also been found to be expressed in the respiratory system and, in order to examine the function of beta defensins in vivo, gene targeting was used to generate Defb1-deficient (Defb1(tm1Hgu)/Defb1(tm1Hgu) [Defb1(-/-)]) mice. The Defb1 synthetic peptide was shown to have a salt-sensitive antimicrobial activity that was stronger against Staphylococcus aureus than against Escherichia coli or Pseudomonas aeruginosa.
View Article and Find Full Text PDFThe majority of cystic fibrosis patients produce a mutant form of CFTR (DeltaF508) which has been shown to be mislocalized in both humans and mice. G480C, another clinically 'severe' mutation, has also been demonstrated to be defective in its intracellular processing, but when allowed to traffic in Xenopus oocytes showed similar channel characteristics to that of wild-type CFTR. We have replicated the G480C mutation in the murine Cftr gene using the 'hit and run' double recombination procedure.
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