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At least 793 records · Page 44Linked to original sources
PubMed2004

Nitrous oxide (N(2)O) requires the N-methyl-D-aspartate receptor for its action in Caenorhabditis elegans.

Nitrous oxide (N(2)O, also known as laughing gas) and volatile anesthetics (VAs), the original and still most widely used general anesthetics, produce anesthesia by ill-defined mechanisms. Electrophysiological experiments in vertebrate neurons have suggested that N(2)O and VAs may act by distinct mechanisms; N(2)O antagonizes the N-methyl-d-aspartate (NMDA) subtype of glutamate receptors, whereas VAs alter the function of a variety of other synaptic proteins. However, no genetic or pharmacological experiments have demonstrated that any of these in vitro actions are responsible for the behavioral effects of either class of anesthetics. By using genetic tools in Caenorhabditis elegans, we tested whether the action of N(2)O requires the NMDA receptor in vivo and whether its mechanism is shared by VAs. Distinct from the action of VAs, N(2)O produced behavioral defects highly specific and characteristic of that produced by loss-of-function mutations in both NMDA and non-NMDA glutamate receptors. A null mutant of nmr-1, which encodes a C. elegans NMDA receptor, was completely resistant to the behavioral effects of N(2)O, whereas a non-NMDA receptor-null mutant was normally sensitive. The N(2)O-resistant nmr-1(null) mutant was not resistant to VAs. Likewise, VA-resistant mutants had wild-type sensitivity to N(2)O. Thus, the behavioral effects of N(2)O require the NMDA receptor NMR-1, consistent with the hypothesis formed from vertebrate electrophysiological data that a major target of N(2)O is the NMDA receptor.

Acetylcholine↗
PubMed2004

Gamma protocadherins are required for synaptic development in the spinal cord.

Fifty-eight cadherin-related protocadherin (Pcdh) genes are tandemly arrayed in three clusters (alpha, beta, and gamma) on mouse chromosome 18. The large number of clustered Pcdh family members, their presence at synapses, and the known binding specificities of other cadherin superfamily members all suggest that these Pcdhs play roles in specifying synaptic connectivity. Consistent with this idea, mice lacking all 22 genes of the Pcdh-gamma cluster have decreased numbers of spinal cord synapses and are nearly immobile. Interpretation of this phenotype was complicated, however, by the fact that Pcdh-gamma loss also led to apoptosis of many spinal interneurons. Here, we used two methods to circumvent apoptosis and neurodegeneration in Pcdh-gamma mutant mice. First, we analyzed mutants lacking both Pcdh-gamma proteins and the proapoptotic protein Bax. Second, we generated a hypomorphic allele of Pcdh-gamma in which apoptosis was minimal. In both cases, spinal interneurons were preserved but the mice bore dramatically decreased numbers of spinal cord synapses and exhibited profound neurological defects. Moreover, synaptic function was compromised in neurons cultured from the hypomorphs. These results provide evidence for a direct role of gamma-Pcdhs in synaptic development and establish genetic tools for elucidating their contribution to synaptic specificity.

Animals↗
PubMed2002

Mutagenesis of a functional chimeric gene in yeast identifies mutations in the simian virus 40 large T antigen J domain.

Simian virus 40 large T antigen contains an amino terminal J domain that catalyzes T antigen-mediated viral DNA replication and cellular transformation. To dissect the role of the J domain in these processes, we exploited the genetic tools available only in the yeast Saccharomyces cerevisiae to isolate 14 loss-of-function point mutations in the T antigen J domain. This screen also identified mutations that, when engineered into simian virus 40, resulted in T antigen mutants that were defective for the ability to support viral growth, to transform mammalian cells in culture, to dissociate the p130-E2F4 transcription factor complex, and to stimulate ATP hydrolysis by hsc70, a hallmark of J domain-containing molecular chaperones. These data correlate the chaperone activity of the T antigen J domain with its roles in viral infection and cellular transformation and support a model by which the viral J domain recruits the cytoplasmic hsc70 molecular chaperone in the host to rearrange multiprotein complexes implicated in replication and transformation. More generally, this study presents the use of a yeast screen to identify loss-of-function mutations in a mammalian virus and can serve as a widely applicable method to uncover domain functions of mammalian proteins for which there are yeast homologues with selectable mutant phenotypes.

Adenosine Triphosphate↗
PubMed2005

High-frequency gene targeting in Arabidopsis plants expressing the yeast RAD54 gene.

Gene targeting, which is homologous recombination-mediated integration of an extra-chromosomal DNA segment into a chromosomal target sequence, enables the precise disruption or replacement of any gene. Despite its value as a molecular genetic tool, gene targeting remains an inefficient technology in most species. We report that expression of the yeast RAD54 gene, a member of the SWI2/SNF2 chromatin remodeling gene family, enhances gene targeting in Arabidopsis by one to two orders of magnitude, from 10(-4) to 10(-3) in WT plants to 10(-2) to 10(-1). We show that integration events, detected with an assay based on the use of a fluorescent seed marker, are precise and germinally transmitted. These findings suggest that chromatin remodeling is rate-limiting for gene targeting in plants and improves the prospects for using gene targeting for the precise modification of plant genomes.

Agrobacterium tumefaciens↗
PubMed2005

Retrograde signaling from the brain to the retina modulates the termination of the light response in Drosophila.

A critical factor in visual function is the speed with which photoreceptors (PRs) return to the resting state when light intensity dims. Several elements subserve this process, many of which promote the termination of the phototransduction cascade. Although the known elements are intrinsic to PRs, we have found that prompt restoration to the resting state of the Drosophila electroretinogram can require effective communication between the retina and the underlying brain. The requirement is seen more dramatically with long than with short light pulses, distinguishing the phenomenon from gross disruption of the termination machinery. The speed of recovery is affected by mutations (in the Hdc and ort genes) that prevent PRs from transmitting visual information to the brain. It is also affected by manipulation (using either drugs like neostigmine or genetic tools to inactivate neurotransmitter release) of cholinergic signals that arise in the brain. Intracellular recordings support the hypothesis that PRs are the target of this communication. We infer that signaling from the retina to the optic lobe prompts a feedback signal to retinal PRs. Although the mechanism of this retrograde signaling remains to be discerned, the phenomenon establishes a previously unappreciated mode of control of the temporal responsiveness of a primary sensory neuron.

Animals↗
PubMed2002

Neuronal activity-dependent membrane traffic at the neuromuscular junction.

During development and also in adulthood, synaptic connections are modulated by neuronal activity. To follow such modifications in vivo, new genetic tools are designed. The nontoxic C-terminal fragment of tetanus toxin (TTC) fused to a reporter gene such as LacZ retains the retrograde and transsynaptic transport abilities of the holotoxin itself. In this work, the hybrid protein is injected intramuscularly to analyze in vivo the mechanisms of intracellular and transneuronal traffics at the neuromuscular junction (NMJ). Traffic on both sides of the synapse are strongly dependent on presynaptic neural cell activity. In muscle, a directional membrane traffic concentrates beta-galactosidase-TTC hybrid protein into the NMJ postsynaptic side. In neurons, the probe is sorted across the cell to dendrites and subsequently to an interconnected neuron. Such fusion protein, sensitive to presynaptic neuronal activity, would be extremely useful to analyze morphological changes and plasticity at the NMJ.

Animals↗
PubMed2000

A sensitized genetic system for the analysis of murine B lymphocyte signal transduction pathways dependent on Bruton's tyrosine kinase.

Modifier screens have been powerful genetic tools to define signaling pathways in lower organisms. The identification of modifier loci in mice has begun to allow a similar dissection of mammalian signaling pathways. Transgenic mice (Btk(lo)) expressing 25% of endogenous levels of Bruton's tyrosine kinase (Btk) have B cell functional responses between those of wild-type and Btk(-/-) mice. We asked whether reduced dosage or complete deficiency of genes previously implicated as Btk regulators would modify the Btk(lo) phenotype. We used two independent assays of Btk-dependent B cell function. Proliferative response to B cell antigen receptor cross-linking in vitro was chosen as an example of a relatively simple, well-defined signaling system. In vivo response to type II T-independent antigens (TI-II) measures complex interactions among multiple cell types over time and may identify additional Btk pathways. All modifiers identified differentially affected these two assays, indicating that Btk mediates these processes via distinct mechanisms. Loss of Lyn, PTEN (phosphatase and tensin homolog), or SH2-containing inositol phosphatase suppressed the Btk(lo) phenotype in vitro but not in vivo, whereas CD19 and the p85alpha form of phosphoinositide 3-kinase behaved as Btk(lo) enhancers in vivo but not in vitro. Effects of Lyn, PTEN, or p85alpha haploinsufficiency were observed. Haploinsufficiency or complete deficiency of protein kinase C beta, Fyn, CD22, Galphaq, or Galpha11 had no detectable effect on the function of Btk(lo) B cells. A transgenic system creating a reduction in dosage of Btk can therefore be used to identify modifier loci that affect B cell responses and quantitatively rank their contribution to Btk-mediated processes.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1987

Transposition of bacteriophage Mu in the Legionnaires disease bacterium.

Legionnaires disease is an acute respiratory disease that is often fatal for immunocompromised patients. The causative agent of this disease, Legionella pneumophila, is a Gram-negative bacterium that is present in a variety of aquatic environments. L. pneumophila is a facultative intracellular parasite; it grows within human phagocytic cells and eventually causes their destruction. In contrast to many other intracellular parasites, L. pneumophila is a Gram-negative bacterium that can be grown in standard microbiological culture medium. To determine the factors that enable this organism to enter, survive, and multiply within human mononuclear phagocytes, we chose bacteriophage Mu, a powerful genetic tool that transposes within the host cell genome, to generate insertion mutations and gene fusions in the Legionella genome. Certain derivatives of Mu are able to generate fusions between target genes and the lac operon from Escherichia coli. We have determined that although Mu is unable to attach to L. pneumophila or complete its life cycle within Legionella, it does transpose within the Legionella genome. Transposition was detected with a mini-Mu phage that carries the lac operon of E. coli.

Bacteriophage mu↗
PubMed1997

Phylogeny of the genus Pistacia as determined from analysis of the chloroplast genome.

Classification within the genus Pistacia has been based on leaf morphology and geographical distribution. Molecular genetic tools (PCR amplification followed by restriction analysis of a 3.2-kb region of variable chloroplast DNA, and restriction fragment length polymorphism analysis of the Pistacia cpDNA with tobacco chloroplast DNA probes) provided a new set of variables to study the phylogenetic relationships of 10 Pistacia species. Both parsimony and cluster analyses were used to divide the genus into two major groups. P. vera was determined to be the least derived species. P. weinmannifolia, an Asian species, is most closely related to P. texana and P. mexicana, New World species. These three species share a common origin, suggesting that a common ancestor of P. texana and P. mexicana originated in Asia. P. integerrima and P. chinensis were shown to be distinct whereas the pairs of species were monophyletic within each of two tertiary groups, P. vera:P. khinjuk and P. mexicana:P. texana. An evolutionary trend from large to small nuts and leaves with few, large leaflets to many, small leaflets was supported. The genus Pistacia was shown to have a low chloroplast DNA mutation rate: 0.05-0.16 times that expected of annual plants.

Chloroplasts↗
PubMed1998

Tn552 transposase catalyzes concerted strand transfer in vitro.

The Tn552 transposase, a member of the DDE superfamily of transposase and retroviral integrase proteins, has been expressed in soluble form. The purified protein performs concerted strand transfer in vitro, efficiently pairing two preprocessed transposon ends and inserting them into target DNA. For maximum efficiency, both participating DNA ends must contain the two adjacent transposase-binding sites that are the normal constituents of the Tn552 termini. As is the case with transposition in vivo, the insertions recovered from the reaction in vitro are flanked by repeats of a short target sequence, most frequently 6 bp. The reaction has stringent requirements for a divalent metal ion. Concerted strand transfer is most efficient with Mg2+. Although it stimulates strand transfer overall, Mn2+ promotes uncoupled, single-ended events at the expense of concerted insertions. The simplicity and efficiency of the Tn552 transposition system make it an attractive subject for structural and biochemical studies and a potentially useful genetic tool.

Base Sequence↗
PubMed1999

Stimulation of homologous recombination in plants by expression of the bacterial resolvase ruvC.

Targeted gene disruption exploits homologous recombination (HR) as a powerful reverse genetic tool, for example, in bacteria, yeast, and transgenic knockout mice, but it has not been applied to plants, owing to the low frequency of HR and the lack of recombinogenic mutants. To increase the frequency of HR in plants, we constructed transgenic tobacco lines carrying the Escherichia coli RuvC gene fused to a plant viral nuclear localization signal. We show that RuvC, encoding an endonuclease that binds to and resolves recombination intermediates (Holliday junctions) is properly transcribed in these lines and stimulates HR. We observed a 12-fold stimulation of somatic crossover between genomic sequences, a 11-fold stimulation of intrachromosomal recombination, and a 56-fold increase for the frequency of extrachromosomal recombination between plasmids cotransformed into young leaves via particle bombardment. This stimulating effect may be transferred to any plant species to obtain recombinogenic plants and thus constitutes an important step toward gene targeting.

Journal Article↗
PubMed2000

Rapid generation of nested chromosomal deletions on mouse chromosome 2.

Nested chromosomal deletions are powerful genetic tools. They are particularly suited for identifying essential genes in development either directly or by screening induced mutations against a deletion. To apply this approach to the functional analysis of mouse chromosome 2, a strategy for the rapid generation of nested deletions with Cre recombinase was developed and tested. A loxP site was targeted to the Notch1 gene on chromosome 2. A targeted line was cotransfected with a second loxP site and a plasmid for transient expression of Cre. Independent random integrations of the second loxP site onto the targeted chromosome in direct repeat orientation created multiple nested deletions. By virtue of targeting in an F(1) hybrid embryonic stem cell line, F(1)(129S1xCast/Ei), the deletions could be verified and rapidly mapped. Ten deletions fell into seven size classes, with the largest extending six or seven centiMorgans. The cytology of the deletion chromosomes were determined by fluorescent in situ hybridization. Eight deletions were cytologically normal, but the two largest deletions had additional rearrangements. Three deletions, including the largest unrearranged deletion, have been transmitted through the germ line. Several endpoints also have been cloned by plasmid rescue. These experiments illustrate the means to rapidly create and map deletions anywhere in the mouse genome. They also demonstrate an improved method for generating nested deletions in embryonic stem cells.

Animals↗
PubMed1998

Deletion of the Saccharomyces cerevisiae TRR1 gene encoding thioredoxin reductase inhibits p53-dependent reporter gene expression.

The prevalence of p53 gene mutations in many human tumors implies that p53 protein plays an important role in preventing cancers. Central among the activities ascribed to p53 is its ability to stimulate transcription of other genes that inhibit cells from entering S phase with damaged DNA. Human p53 can be studied in yeast where genetic tools can be used to identify proteins that affect its ability to stimulate transcription. Although p53 strongly stimulated reporter gene expression in wild type yeast, it only weakly stimulated reporter gene expression in Deltatrr1 yeast that lacked the gene encoding thioredoxin reductase. Furthermore, ectoptic expression of TRR1 in Deltatrr1 yeast restored p53-dependent reporter gene activity to high levels. Immunoblot assays established that the Deltatrr1 mutation affected the activity and not the level of p53 protein. The results suggest that p53 can form disulfides and that these disulfides must be reduced in order for the protein to function as a transcription factor.

DNA-Binding Proteins↗
PubMed1999

Functional reconstitution of human telomerase expressed in Saccharomyces cerevisiae.

Telomerase is a ribonucleoprotein enzyme complex that adds DNA repeats at the ends of chromosomes. In an effort to establish an in vivo heterologous expression system for active human telomerase, we expressed human telomerase reverse transcriptase (hTERT) in Saccharomyces cerevisiae and affinity-purified the protein as a fusion with glutathione S-transferase (GST). Addition of the GST moiety to the N terminus of hTERT did not interfere with telomerase activity when GST-hTERT was expressed in rabbit reticulocyte lysate (RRL) in the presence of the human telomerase RNA (hTR). Active human telomerase was immunoprecipitated from yeast lysates that co-expressed GST-hTERT and hTR. In addition, telomerase activity could be reconstituted in vitro by the addition of hTR to GST-hTERT that was immunoprecipitated from either RRL or S. cerevisiae lysates. The expression and reconstitution of human telomerase activity in yeast will provide powerful biochemical and genetic tools to study the various components required for the assembly and function of this enzyme.

Base Sequence↗
PubMed2003

Functional characterization of Drosophila sialyltransferase.

Sialylation is an important carbohydrate modification of glycoconjugates in the deuterostome lineage of animals. By contrast, the evidence for sialylation in protostomes has been scarce and somewhat controversial. In the present study, we characterize a Drosophila sialyltransferase gene, thus providing experimental evidence for the presence of sialylation in protostomes. This gene encodes a functional alpha2-6-sialyltransferase (SiaT) that is closely related to the vertebrate ST6Gal sialyltransferase family, indicating an ancient evolutionary origin for this family. Characterization of recombinant, purified Drosophila SiaT revealed a novel acceptor specificity as it exhibits highest activity toward GalNAcbeta1-4GlcNAc carbohydrate structures at the non-reducing termini of oligosaccharides and glycoprotein glycans. Oligosaccharides are preferred over glycoproteins as acceptors, and no activity toward glycolipid acceptors was detected. Recombinant Drosophila SiaT expressed in cultured insect cells possesses in vivo and in vitro autosialylation activity toward beta-linked GalNAc termini of its own N-linked glycans, thus representing the first example of a sialylated insect glycoconjugate. In situ hybridization revealed that Drosophila SiaT is expressed during embryonic development in a tissue- and stage-specific fashion, with elevated expression in a subset of cells within the central nervous system. The identification of a SiaT in Drosophila provides a new evolutionary perspective for considering the diverse functions of sialylation and, through the powerful genetic tools available in this system, a means of elucidating functions for sialylation in protostomes.

Amino Acid Sequence↗
PubMed2004

Transposon mutagenesis of Trypanosoma brucei identifies glycosylation mutants resistant to concanavalin A.

We have engineered Trypanosoma brucei with a novel mariner transposition system that allows large populations of mutant cells to be generated and screened. As a proof of principle, we isolated and characterized two independent clones that were resistant to the cytotoxic action of concanavalin A. In both clones, the transposon had integrated into the locus encoding a homologue of human ALG12, which encodes a dolichyl-P-Man: Man(7)GlcNAc(2)-PP-dolichyl-alpha6-mannosyltransferase. Conventional knock-out of ALG12 in a wild-type background gave an identical phenotype to the mariner mutants, and biochemical analysis confirmed that they have the same defect in the N-linked oligosaccharide synthesis pathway. To our surprise, both mariner mutants were homozygous; the second allele appeared to have undergone gene conversion by the mariner-targeted allele. Subsequent experiments showed that the frequency of gene conversion at the ALG12 locus, in the absence of selection, was 0.25%. As we approach the completion of the trypanosome genome project, transposon mutagenesis provides an important addition to the repertoire of genetic tools for T. brucei.

Amino Acid Sequence↗
PubMed2007

Regulation of Nanog expression by phosphoinositide 3-kinase-dependent signaling in murine embryonic stem cells.

Embryonic stem (ES) cell pluripotency is regulated by a combination of extrinsic and intrinsic factors. Previously we have demonstrated that phosphoinositide 3-kinase (PI3K)-dependent signaling is required for efficient self-renewal of murine ES cells. In the study presented here, we have investigated the downstream molecular mechanisms that contribute to the ability of PI3Ks to regulate pluripotency. We show that inhibition of PI3K activity with either pharmacological or genetic tools results in decreased expression of RNA for the homeodomain transcription factor Nanog and decreased Nanog protein levels. Inhibition of glycogen synthase kinase 3 (GSK-3) activity by PI3Ks plays a key role in regulation of Nanog expression, because blockade of GSK-3 activity effectively reversed the effects of PI3K inhibition on Nanog RNA, and protein expression and self-renewal under these circumstances were restored. Furthermore, GSK-3 mutants mimicked the effects of PI3K or GSK-3 inhibition on Nanog expression. Importantly, expression of an inducible form of Nanog prevented the loss of self-renewal observed upon inhibition of PI3Ks, supporting a functional relationship between PI3Ks and Nanog expression. In addition, expression of a number of putative Nanog target genes was sensitive to PI3K inhibition. Thus, the new evidence provided in this study shows that PI3K-dependent regulation of ES cell self-renewal is mediated, at least in part, by the ability of PI3K signaling to maintain Nanog expression. Regulation of GSK-3 activity by PI3Ks appears to play a key role in this process.

Animals↗
PubMed2001

Invasion history and genetic population structure of riverine macroinvertebrates.

Macroinvertebrate communities of large rivers have experienced dramatic species turnovers in the last decades, which still go on. The analysis of genetic population structure plays a central role in understanding and predicting these biological invasions. Two points of view are considered: the influence of the invasion history on the genetic structuring and the potential implications of genetic structure for future invasibility. Expectations about selectively neutral genetic variation in simple invasion models are compared to case studies of amphipods and Dreissena. The genetic patterns of one amphipod species of the Gammarus fossarum complex yield strong evidence for a stepwise regional colonisation. Other invasive amphipods show similar, but less clear genetic characteristics. Long-distance colonisation is probably common in Dreissena polymorpha, but subsequent downstream swamping of larvae obscures the expected genetic pattern by homogenising neighbouring populations. First, a brief review of general characteristics of riverine invasions is followed by a discussion of the utility of genetic tools for inferring taxon and source population identity.

Journal Article↗