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At least 883 records · Page 49Linked to original sources
PubMed2004

Short interfering RNA-mediated inhibition of herpes simplex virus type 1 gene expression and function during infection of human keratinocytes.

RNA interference (RNAi) is an antiviral mechanism that is activated when double-stranded RNA is cleaved into fragments, called short interfering RNA (siRNA), that prime an inducible gene silencing enzyme complex. We applied RNAi against a herpes simplex virus type 1 (HSV-1) gene, glycoprotein E, which mediates cell-to-cell spread and immune evasion. In an in vitro model of infection, human keratinocytes were transfected with siRNA specific for glycoprotein E and then infected with wild-type HSV-1. RNAi-mediated gene silencing reproduced the small plaque phenotype of a gE-deletion mutant virus. The specificity of gene targeting was demonstrated by flow cytometry and Northern blot analyses. Exogenous siRNA can suppress HSV-1 glycoprotein E expression and function during active infection in vitro through RNAi. This work establishes RNAi as a genetic tool for the study of HSV and provides a foundation for development of RNAi as a novel antiviral therapy.

Blotting, Northern↗
PubMed2001

Sequence-specific recognition and cleavage of telomeric repeat (TTAGG)(n) by endonuclease of non-long terminal repeat retrotransposon TRAS1.

The telomere of the silkworm Bombyx mori consists of (TTAGG/CCTAA)(n) repeats and harbors a large number of telomeric repeat-specific non-long terminal repeat retrotransposons, such as TRAS1 and SART1. To understand how these retrotransposons recognize and integrate into the telomeric repeat in a sequence-specific manner, we expressed the apurinic-apryrimidinic endonuclease-like endonuclease domain of TRAS1 (TRAS1 EN), which is supposed to digest the target DNA, and characterized its enzymatic properties. Purified TRAS1 EN could generate specific nicks on both strands of the telomeric repeat sequence between T and A of the (TTAGG)(n) strand (bottom strand) and between C and T of the (CCTAA)(n) strand (top strand). These sites are consistent with insertion sites expected from the genomic structure of boundary regions of TRAS1. Time course studies of nicking activities on both strands revealed that the cleavages on the bottom strand preceded those on the top strand, supporting the target-primed reverse transcription model. TRAS1 EN could cleave the telomeric repeats specifically even if it was flanked by longer tracts of nontelomeric sequence, indicating that the target site specificity of the TRAS1 element was mainly determined by its EN domain. Based on mutation analyses, TRAS1 EN recognizes less than 10 bp around the initial cleavage site (upstream 7 bp and downstream 3 bp), and the GTTAG sequence especially is essential for the cleavage reaction on the bottom strand (5'. TTAGGTT downward arrow AGG. 3'). TRAS1 EN, the first identified endonuclease digesting telomeric repeats, may be used as a genetic tool to shorten the telomere in insects and some other organisms.

Amino Acid Sequence↗
PubMed2004

Signaling in glial development: differentiation migration and axon guidance.

Glial cells have diverse functions that are necessary for the proper development and function of complex nervous systems. During development, a variety of reciprocal signaling interactions between glia and neurons dictate all parts of nervous system development. Glia may provide attractive, repulsive, or contact-mediated cues to steer neuronal growth cones and ensure that neurons find their appropriate synaptic targets. In fact, both neurons and glia may act as migrational substrates for one another at different times during development. Also, the exchange of trophic signals between glia and neurons is essential for the proper bundling, fasciculation, and ensheathement of axons as well as the differentiation and survival of both cell types. The growing number of links between glial malfunction and human disease has generated great interest in glial biology. Because of its relative simplicity and the many molecular genetic tools available, Drosophila is an excellent model organism for studying glial development. This review will outline the roles of glia and their interactions with neurons in the embryonic nervous system of the fly.

Animals↗
PubMed1996

Heterocyst formation.

Heterocysts are microaerobic, N2-fixing cells that form in a patterned array within O2-producing filamentous cyanobacteria. Structural features of heterocysts can be predicted from consideration of their physiology. This review focuses on the spacing mechanism that determines which cells will differentiate, and on the regulation of the progression of the differentiation process. Applicable genetic tools, developed primarily using Anabaena PCC 7120, but employed also with Nostoc spp., are reviewed. These tools include localization of transcription using fusions to lux, lac, and gfp, and mutagenesis with oriV-containing derivatives of transposon Tn5. Mature and developing heterocysts inhibit nearby vegetative cells from differentiating; genes patA, devA, hetC, and the hetMNI locus may hold keys to understanding intercellular interactions that influence heterocyst formation. Regulatory and other genes that are transcriptionally activated at different times after nitrogen stepdown have been identified, and should permit analysis of mechanisms that underlie the progression of heterocyst differentiation.

Anabaena↗
PubMed1999

Plant retrotransposons.

Retrotransposons are mobile genetic elements that transpose through reverse transcription of an RNA intermediate. Retrotransposons are ubiquitous in plants and play a major role in plant gene and genome evolution. In many cases, retrotransposons comprise over 50% of nuclear DNA content, a situation that can arise in just a few million years. Plant retrotransposons are structurally and functionally similar to the retrotransposons and retroviruses that are found in other eukaryotic organisms. However, there are important differences in the genomic organization of retrotransposons in plants compared to some other eukaryotes, including their often-high copy numbers, their extensively heterogeneous populations, and their chromosomal dispersion patterns. Recent studies are providing valuable insights into the mechanisms involved in regulating the expression and transposition of retrotransposons. This review describes the structure, genomic organization, expression, regulation, and evolution of retrotransposons, and discusses both their contributions to plant genome evolution and their use as genetic tools in plant biology.

Evolution, Molecular↗
PubMed2004

The cellulosomes: multienzyme machines for degradation of plant cell wall polysaccharides.

The discrete multicomponent, multienzyme cellulosome complex of anaerobic cellulolytic bacteria provides enhanced synergistic activity among the different resident enzymes to efficiently hydrolyze intractable cellulosic and hemicellulosic substrates of the plant cell wall. A pivotal noncatalytic subunit called scaffoldin secures the various enzymatic subunits into the complex via the cohesin-dockerin interaction. The specificity characteristics and tenacious binding between the scaffoldin-based cohesin modules and the enzyme-borne dockerin domains dictate the supramolecular architecture of the cellulosome. The diversity in cellulosome architecture among the known cellulosome-producing bacteria is manifest in the arrangement of their genes in either multiple-scaffoldin or enzyme-linked clusters on the genome. The recently described three-dimensional crystal structure of the cohesin-dockerin heterodimer sheds light on the critical amino acids that contribute to this high-affinity protein-protein interaction. In addition, new information regarding the regulation of cellulosome-related genes, budding genetic tools, and emerging genomics of cellulosome-producing bacteria promises new insight into the assembly and consequences of the multienzyme complex.

Amino Acid Sequence↗
PubMed2006

Defining virulence genes in the dimorphic fungi.

Most dimorphic fungal pathogens cause respiratory disease in mammals and must therefore possess virulence mechanisms to combat and overcome host pulmonary defenses. Over the past decade, advances in genetic tools have made it possible to investigate the basis of dimorphic fungal pathogenesis at the molecular level. Gene disruptions and RNA interference have now formally demonstrated the involvement of six virulence factors: CBP, alpha-(1,3)-glucan, BAD1, SOWgp, Mep1, and urease. Additional candidate virulence-associated genes have been identified on the premise that factors necessary for pathogenicity are associated specifically with the parasitic form. This principle continues to form the foundation for genomics-based analyses to further augment the list. Thus, the stage is set and the tools are in place for the next phase of medical mycology research: defining the virulence-associated factors underlying the success of dimorphic fungal pathogens.

Arginine↗
PubMed2000

5-ht6 receptors as emerging targets for drug discovery.

5-ht6 receptors are the latest serotonin receptors to be identified by molecular cloning. Their high affinity for a wide range of drugs used in psychiatry, coupled with their intriguing distribution in the brain, has stimulated significant interest. Antisense oligonucleotides, antipeptide antibodies, selective radioligands, knockout mice, and selective antagonists of the 5-ht6 receptor have recently become available. Surprisingly, 5-ht6 receptors appear to regulate cholinergic neurotransmission in the brain, rather than the expected interaction as modulators of dopaminergic transmission. This interaction predicts a possible role for 5-ht6 receptor antagonists in the treatment of learning and memory disorders. Furthermore, polymorphisms in the sequence of the 5-ht6 receptor gene may provide a genetic tool to further our understanding of the differential responses of patients to antipsychotic medications.

Animals↗
PubMed2003

Spectrotemporal structure of receptive fields in areas AI and AAF of mouse auditory cortex.

The mouse is a promising model system for auditory cortex research because of the powerful genetic tools available for manipulating its neural circuitry. Previous studies have identified two tonotopic auditory areas in the mouse-primary auditory cortex (AI) and anterior auditory field (AAF)- but auditory receptive fields in these areas have not yet been described. To establish a foundation for investigating auditory cortical circuitry and plasticity in the mouse, we characterized receptive-field structure in AI and AAF of anesthetized mice using spectrally complex and temporally dynamic stimuli as well as simple tonal stimuli. Spectrotemporal receptive fields (STRFs) were derived from extracellularly recorded responses to complex stimuli, and frequency-intensity tuning curves were constructed from responses to simple tonal stimuli. Both analyses revealed temporal differences between AI and AAF responses: peak latencies and receptive-field durations for STRFs and first-spike latencies for responses to tone bursts were significantly longer in AI than in AAF. Spectral properties of AI and AAF receptive fields were more similar, although STRF bandwidths were slightly broader in AI than in AAF. Finally, in both AI and AAF, a substantial minority of STRFs were spectrotemporally inseparable. The spectrotemporal interaction typically appeared in the form of clearly disjoint excitatory and inhibitory subfields or an obvious spectrotemporal slant in the STRF. These data provide the first detailed description of auditory receptive fields in the mouse and suggest that although neurons in areas AI and AAF share many response characteristics, area AAF may be specialized for faster temporal processing.

Acoustic Stimulation↗
PubMed2004

Strains of rodents and the pharmacology of learning and memory.

Mendelian genetic tools have extensively been used to improve the description of the pharmacological mechanisms involved in learning and memory. The first part of this short review describes experiments involving the bidirectional selection of rats or mice for extreme behavioral characteristics or for sensitivity to pharmacological treatments. The second part focuses specifically on in-breeding. In conclusion, the advantages and the limits of a Mendelian pharmacogenetic approach of learning and memory are discussed.

Animals↗
PubMed1996

Assignment of the human tryptophanyl-tRNA synthetase gene (WARS) to chromosome 14q32.2 --> q32.32.

Tryptophanyl-tRNA synthetase catalyzes the aminoacylation of tRNAtrp with tryptophan, an essential function in the cell's protein synthesis machinery. It has been shown that tryptophanyl-tRNA synthetase is induced by interferon, and this has led to hypotheses about other possible functions of tryptophanyl-tRNA synthetase. We have mapped a cDNA probe of the tryptophanyl-tRNA synthetase gene (WARS) by a combination of somatic cell hybrid analysis, fluorescence in situ hybridization (FISH), and linkage analysis. Both FISH and linkage analysis independently supported a more distal position of WARS than had been previously reported. FISH mapping indicated a most likely location at 14q32.31. Linkage analysis was based on the 40 reference families from the CEPH collaboration and resulted in a 13-point map, placing WARS, with odds of more than 1,000:1, within an area of approximately 10 cM and, with odds of 198:1, in an approximately 6 cM interval between pCMM101 and D14S27. The study provides additional integration of the physical and genetic maps of the distal part of 14q, as well as genetic tools enabling a more complete understanding of the function of tryptophanyl-tRNA synthetase, especially with regard to the cryptic inducibility of interferon.

Chromosome Mapping↗
PubMedDate not supplied

Atherosclerosis susceptibility differences among progenitors of recombinant inbred strains of mice.

Female mice of 16 inbred mouse strains were fed an atherogenic diet for 14 weeks and were then evaluated for atherosclerotic lesions in the aorta. Strains C57BL/6, C57BR/cd, C57L, and SM were very susceptible to atherosclerosis, with lesion area/aortic cross-sections in the range of 4500 to 8000 microns 2. Strains C58 and SWR were intermediate in susceptibility, with lesion area/sections in the range of 1670 to 1690 microns 2. Strains 129, AKR, DBA/2, and BALB/c had only small lesions in the range of 20 to 350 microns 2/section; strains C3H, NZB, CBA, HRS, SJL, and A had no lesions after 14 weeks. Lesion formation in five strains was compared at several time points. Strain C57BL/6 mice developed lesions by 7 weeks, and these continued to grow until all mice had large atheromatous plaques in the aorta and coronary arteries. Strains AKR and DBA/2 also had fatty streak lesions as early as 7 or 8 weeks, but these lesions had not progressed in size by 14 weeks. Strains BALB/c and C3H, which were both resistant to lesion formation at 14 weeks, diverged from each other as time progressed. By 1 year, BALB/c mice had large lesions, but C3H mice had none. Most of the inbred strains chosen for evaluation are the progenitors of recombinant inbred sets of strains, a genetic tool that greatly facilitates the analysis of strain differences. This survey indicates seven additional recombinant inbred sets of strains whose progenitors differ in atherosclerosis susceptibility: BXD, AKXL, SWXJ, NX8, 129XB, NXSM, and B6NXAKRN. An analysis of these recombinant inbred strains may reveal additional mouse genes affecting atherosclerosis susceptibility.

Animals↗
PubMed2004

Transient receptor potential melastatin 7 ion channels regulate magnesium homeostasis in vascular smooth muscle cells: role of angiotensin II.

Magnesium modulates vascular smooth muscle cell (VSMC) function. However, molecular mechanisms regulating VSMC Mg2+ remain unknown. Using biochemical, pharmacological, and genetic tools, the role of transient receptor potential membrane melastatin 7 (TRPM7) cation channel in VSMC Mg2+ homeostasis was evaluated. Rat, mouse, and human VSMCs were studied. Reverse transcriptase polymerase chain reaction and immunoblotting demonstrated TRPM7 presence in VSMCs (membrane and cytosol). Angiotensin II (Ang II) and aldosterone increased TRPM7 expression. Gene silencing using small interfering RNA (siRNA) against TRPM7, downregulated TRPM7 (mRNA and protein). Basal [Mg2+]i, measured by mag fura-2AM, was reduced in siRNA-transfected cells (0.39+/-0.01 mmol/L) versus controls (0.54+/-0.01 mmol/L; P<0.01). Extracellular Mg2+ dose-dependently increased [Mg2+]i in control cells (Emax 0.70+/-0.02 mmol/L) and nonsilencing siRNA-transfected cells (Emax 0.71+/-0.04 mmol/L), but not in siRNA-transfected cells (Emax 0.5+/-0.01 mmol/L). The functional significance of TRPM7 was evaluated by assessing [Mg2+]i and growth responses to Ang II in TRPM7 knockdown cells. Acute Ang II stimulation decreased [Mg2+]i in control and TRPM7-deficient cells in a Na+-dependent manner. Chronic stimulation increased [Mg2+]i in control, but not in siRNA-transfected VSMCs. Ang II-induced DNA and protein synthesis, measured by 3[H]-thymidine and 3[H]-leucine incorporation, respectively, were increased in control and nonsilencing cells, but not in TRPM7 knockdown VSMCs. Our data indicate that VSMCs possess membrane-associated, Ang II-, and aldosterone-regulated TRPM7 channels, which play a role in regulating basal [Mg2+]i, transmembrane Mg2+ transport and DNA and protein synthesis. These novel findings identify TRPM7 as a functionally important regulator of Mg2+ homeostasis and growth in VSMCs.

Aldosterone↗
PubMed1996

Differential gene expression by Pseudomonas aeruginosa during interaction with respiratory mucus.

Pseudomonas aeruginosa is a common respiratory tract pathogen that causes serious infections in patients with cystic fibrosis. A number of putative virulence factors have been characterized in several laboratories, and some have been implicated in human infections, based on criteria such as the phenotype of isolates from infected patients, an immune response to a particular antigenic factor, and the effect of a virulence factor on infectivity in an animal model. We have developed a series of genetic tools to study the selective regulation of expression of P. aeruginosa genes during interactions of the pathogen with host tissues. These tools are based on direct enrichment of bacteria, when a particular promoter is induced or repressed. We have found that interaction of bacteria with mucus from patients with cystic fibrosis results in marked induction of expression of several genes, including one that encodes a lipopolysaccharide biosynthetic enzyme, a gene for a protein responsible for uptake of the ferric pyochelin siderophore, and a new gene homologous with a class of iron-responsive repressors. The tools described here are useful for identification of induced or repressed genes in various animal models of infection or in controlled laboratory conditions that mimic natural infections of humans. Such genes might not be detectable when bacteria are cultured in laboratory conditions, and these tools are therefore useful for general probing of a bacterial genome for genes regulated during different stages of infection.

Animals↗
PubMed1991

Characterization of seven novel mutations of the c-erbA beta gene in unrelated kindreds with generalized thyroid hormone resistance. Evidence for two "hot spot" regions of the ligand binding domain.

Genetic analysis in our laboratory of families with generalized thyroid hormone resistance (GTHR) has demonstrated tight linkage with a locus, c-erbA beta, encoding a nuclear T3 receptor. Three point mutations and two deletions in this locus have previously been reported in affected individuals in unrelated families as potential molecular bases for this disorder. In the present study, we have used direct sequencing of polymerase chain reaction-amplified exons of the c-erbA beta gene to rapidly identify novel point mutations from seven previously uncharacterized kindreds with GTHR. Six single base substitutions and one single base insertion were identified and found to be clustered in two regions of exons 9 and 10 in the ligand binding domain of the receptor: in the distal ligand-binding subdomain L2 and across the juncture of the taui and dimerization subdomains. Reduction of T3-binding affinity in each of four mutations tested as well as segregation of all mutations to clinically affected individuals strongly supports the hypothesis that these changes are the cause of GTHR in these kindreds. In view of the diversity of clinical phenotypes manifested, the distinct topographic clustering of the mutations provides an invaluable genetic tool for the molecular dissection of thyroid receptor function.

Adolescent↗
PubMed2003

Endothelial and nonendothelial sources of PDGF-B regulate pericyte recruitment and influence vascular pattern formation in tumors.

Tumor-infiltrating blood vessels deviate morphologically and biochemically from normal vessels, raising the prospect of selective pharmacological targeting. Current antiangiogenic approaches focus mainly on endothelial cells, but recent data imply that targeting pericytes may provide additional benefits. Further development of these concepts will require deeper insight into mechanisms of pericyte recruitment and function in tumors. Here, we applied genetic tools to decipher the function of PDGF-B and PDGF-Rbeta in pericyte recruitment in a mouse fibrosarcoma model. In tumors transplanted into PDGF-B retention motif-deficient (pdgf-b(ret/ret)) mice, pericytes were fewer and were partially detached from the vessel wall, coinciding with increased tumor vessel diameter and hemorrhaging. Transgenic PDGF-B expression in tumor cells was able to increase the pericyte density in both WT and pdgf-b(ret/ret) mice but failed to correct the pericyte detachment in pdgf-b(ret/ret) mice. Coinjection of exogenous pericytes and tumor cells showed that pericytes require PDGF-Rbeta for recruitment to tumor vessels, whereas endothelial PDGF-B retention is indispensable for proper integration of pericytes in the vessel wall. Our data support the notion that pericytes serve an important function in tumor vessels and highlight PDGF-B and PDGF-Rbeta as promising molecular targets for therapeutic intervention.

Animals↗
PubMed2006

Misbehaving macrophages in the pathogenesis of psoriasis.

Psoriasis is a chronic inflammatory skin disease unique to humans. In this issue of the JCI, 2 studies of very different mouse models of psoriasis both report that macrophages play a key role in inducing psoriasis-like skin disease. Psoriasis is clearly a polygenic, inherited disease of uncontrolled cutaneous inflammation. The debate that currently rages in the field is whether psoriasis is a disease of autoreactive T cells or whether it reflects an intrinsic defect within the skin--or both. However, these questions have proven difficult to dissect using molecular genetic tools. In the current studies, the authors have used 2 different animal models to address the role of macrophages in disease pathogenesis: Wang et al. use a mouse model in which inflammation is T cell dependent, whereas the model used by Stratis et al. is T cell independent (see the related articles beginning on pages 2105 and 2094, respectively). Strikingly, both groups report an important contribution by macrophages, implying that macrophages can contribute to both epithelial-based and T cell-mediated pathways of inflammation.

Animals↗
PubMed2001

Encoding le quattro stagioni within the mammalian brain: photoperiodic orchestration through the suprachiasmatic nucleus.

Within the suprachiasmatic nucleus (SCN) is a pacemaker that not only drives circadian rhythmicity but also directs the circadian organization of photoperiodic (seasonal) timekeeping. Recent evidence using electrophysiological, molecular, and genetic tools now strongly supports this conclusion. Important questions remain regarding the SCN's precise role(s) in the brain's photoperiodic circuits, especially among different species, and the cellular and molecular mechanisms for its photoperiodic "memory." New data suggesting that SCN "clock" genes may also function as "calendar" genes are a first step toward understanding how a photoperiodic clock is built from cycling molecules.

Animals↗