21,147 research outputs found
Gene transfer into mouse lyoma cells by electroporation in high electric fields
No full textNeumann E, Schaefer-Ridder M, Wang Y, Hofschneider PH. Gene transfer into mouse lyoma cells by electroporation in high electric fields. EMBO Journal. 1982;1(7):841-845.Electric impulses (8 kV/cm, 5 microseconds) were found to increase greatly the uptake of DNA into cells. When linear or circular plasmid DNA containing the herpes simplex thymidine kinase (TK) gene is added to a suspension of mouse L cells deficient in the TK gene and the cells are then exposed to electric fields, stable transformants are formed that survive in the HAT selection medium. At 20 degrees C after the application of three successive electric impulses followed by 10 min to allow DNA entry there result 95 (+/- 3) transformants per 10(6) cells and per 1.2 micrograms DNA. Compared with biochemical techniques, the electric field method of gene transfer is very simple, easily applicable, and very efficient. Because the mechanism of DNA transport through cell membranes is not known, a simple physical model for the enhanced DNA penetration into cells in high electric fields is proposed. According to this ' electroporation model' the interaction of the external electric field with the lipid dipoles of a pore configuration induces and stabilizes the permeation sites and thus enhances cross membrane transport
Os isotopic composition of western Aleutian adakites: Implications for the Re/Os of oceanic crust processed through hot subduction zones
No full textConstraining the behaviour of Re and Os during eclogite melting is required to understand the Re and Os budget and 187Os/188Os of recycled slabs produced at warm subduction zones. It is particularly relevant to early Earth history, a period during which slab melting could have prevailed over dehydration due to higher mantle temperatures. There are however currently few constraints on Re and Os mobility during slab melting. Accordingly, we measured Os, Re and 187Os/188Os in primitive submarine lavas (Mg# ˃ 0.6) from the western Aleutian Arc. These include strongly adakitic rocks shown to be derived from eclogite melting (high-Mg# andesite, dacites and rhyodacites), as well as non-adakitic rocks (high-Mg# andesites, basaltic andesites and basalts) with variable sediment and fluid-derived slab contributions for comparison.
The 187Os/188Os of the adakitic and non-adakitic volcanic rocks vary significantly but largely overlap. In both groups, the most radiogenic values occur in samples with the lowest Os concentrations, thus implicating crustal assimilation as the main cause of Os isotope variations. Adakitic and non-adakitic rocks least affected by crustal assimilation have overlapping 187Os/188Os of 0.141–0.149. We show that the source of the adakites is very unlikely to comprise significant eclogite-derived Os, which suggests no or minimal mobilization of Os during eclogite melting. Eclogitic Os is inferred to be retained in sulphides or replacement phases formed upon sulphide breakdown for which Os has high affinity, such as a platinum-group minerals (PGMs). The small Os budget of the adakites is most likely derived from limited reaction with the mantle wedge during ascent. Degassing has reduced Re contents in most samples, but not for end-member adakites (SiO2 > 67% and Sr/Y > 200; n = 4) that were erupted at seafloor depths > 2500 m. These undegassed samples have elevated Re concentrations (0.8–1.5 ppb) that are positively correlated with Sr/Y and so are interpreted to be primary magmatic concentrations resulting from the mobilization of Re from the slab. Re could either be derived from the eclogites or from the serpentinite-derived fluids fluxing eclogites during melting. The former scenario would produce recycled residual crusts with lower Re/Os than in unmelted eclogites while the latter would result in Re/Os ranging from similar to higher than prior to melting. In both cases, the Re/Os and therefore the time-integrated 187Os/188Os of residual crust produced at warm subduction zones involving slab melting are likely to be different from that processed at cooler typical modern subduction zones. Therefore, if slab melting was an important process during the early Earth, the use of Re and Os partitioning in modern subduction zones to model the source of magmas comprising old recycled oceanic crust, such as the HIMU (high μ = 238U/204Pb) ocean island basalts (OIBs), might lead to erroneous interpretations
Lateral gene transfer and the complex distribution of insertions in eukaryotic enolase
No full textInsertions and deletions in protein-coding genes are relatively rare events compared with sequence substitutions because they are more likely to alter the tertiary structure of the protein. For this reason, insertions and deletions which are clearly homologous are considered to be stable characteristics of the proteins where they are found, and their presence and absence has been used extensively to infer large-scale evolutionary relationships and events. Recently, however, it has been shown that the pattern of highly conserved, clearly homologous insertions at positions with no other detectable homoplasy can be incongruent with the phylogeny of the genes or organisms in which they are found. One case where this has been reported is in the enolase genes of apicomplexan parasites and ciliates, which share homologous insertions in a highly conserved region of the gene with the apparently distantly related enolases of plants. Here we explore the distribution of this character in enolase genes from the third major alveolate group, the dinoflagellates, as well as two groups considered to be closely related to alveolates, haptophytes and heterokonts. With these data, all major groups of the chromalveolates are represented, and the distribution of these insertions is shown to be far more complicated than previously believed. The incongruence between this pattern, the known evolutionary relationships between the organisms, and enolase phylogeny itself cannot be explained by any single event or type of event. Instead, the distribution of enolase insertions is more likely the product of several forces that may have included lateral gene transfer, paralogy, and/or recombination. Of these, lateral gene transfer is the easiest to detect and some well-supported cases of eukaryote-to-eukaryote lateral transfer are evident from the phylogeny. [ABSTRACT FROM AUTHOR]Peer reviewedfinal article publishedPhylogenyRecombinationLateral transferParalog
Use of a murine secreted alkaline phosphatase as a non-immunogenic reporter gene in mice: Murine Alkaline Phosphatase as a Reporter Gene
No full textBackground
The development of any vector system as a gene delivery system requires its optimization in vitro and in vivo . Preliminary studies frequently involve the use of a reporter gene, which allows for the rapid and simple assay of vector function through monitoring expression levels of the reporter gene. However, evaluation of vector efficacy can be compromised by immune responses directed against immunogenic reporter proteins.
Methods
We have cloned a murine secreted alkaline phosphatase (mSEAP), and explored its use as a reporter gene in the context of an early region 1 (E1)‐deleted adenovirus (Ad) vector. Studies involved characterization of gene expression in vitro and in vivo , and immunological responses after gene delivery to mice.
Results
In tissue culture, we show that mSEAP is easily measured quantitatively using a sensitive, commercially available chemiluminescent assay, or visualized directly using histological staining. The level of transgene expression from AdmSEAP was similar to that observed for an Ad vector encoding the human placental secreted alkaline phosphatase (hSEAP). After intravenous administration in mice, AdmSEAP continued to express at high levels for the duration of the experiment (1 month), whereas expression from AdhSEAP declined to background levels over the course of the experiment. Although cytotoxic T‐lymphocytes were not detected against either the murine or human SEAP proteins in mice, antibodies were readily detected against the human protein. No antibodies were detected to mSEAP.
Conclusions
Taken together, these data illustrate that mSEAP is a sensitive, non‐immunogenic reporter gene for preclinical mouse studies. Copyright © 2004 John Wiley & Sons, Ltd
Proline-rich tyrosine kinase 2 mediates gonadotropin-releasing hormone signaling to a specific extracellularly regulated kinase-sensitive transcriptional locus in the luteinizing hormone beta-subunit gene
No full textG protein-coupled receptor regulation of gene transcription primarily occurs through the phosphorylation of transcription factors by MAPKs. This requires transduction of an activating signal via scaffold proteins that can ultimately determine the outcome by binding signaling kinases and adapter proteins with effects on the target transcription factor and locus of activation. By investigating these mechanisms, we have elucidated how pituitary gonadotrope cells decode an input GnRH signal into coherent transcriptional output from the LH beta-subunit gene promoter. We show that GnRH activates c-Src and multiple members of the MAPK family, c-Jun NH2-terminal kinase 1/2, p38MAPK, and ERK1/2. Using dominant-negative point mutations and chemical inhibitors, we identified that calcium-dependent proline-rich tyrosine kinase 2 specifically acts as a scaffold for a focal adhesion/cytoskeleton-dependent complex comprised of c-Src, Grb2, and mSos that translocates an ERK-activating signal to the nucleus. The locus of action of ERK was specifically mapped to early growth response-1 (Egr-1) DNA binding sites within the LH beta-subunit gene proximal promoter, which was also activated by p38MAPK, but not c-Jun NH2-terminal kinase 1/2. Egr-1 was confirmed as the transcription factor target of ERK and p38MAPK by blockade of protein expression, transcriptional activity, and DNA binding. We have identified a novel GnRH-activated proline-rich tyrosine kinase 2-dependent ERK-mediated signal transduction pathway that specifically regulates Egr-1 activation of the LH beta-subunit proximal gene promoter, and thus provide insight into the molecular mechanisms required for differential regulation of gonadotropin gene expression
A well-conserved Plasmodium falciparum var gene shows an unusual stage-specific transcript pattern
No full textThe var multicopy gene family encodes Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) variant antigens, which, through their ability to adhere to a variety of host receptors, are thought to be important virulence factors. The predominant expression of a single cytoadherent PfEMP1 type on an infected red blood cell, and the switching between different PfEMP1 types to evade host protective antibody responses, are processes thought to be controlled at the transcriptional level. Contradictory data have been published on the timing of var gene transcription. Reverse transcription-polymerase chain reaction (RT-PCR) data suggested that transcription of the predominant var gene occurs in the later (pigmented trophozoite) stages, whereas Northern blot data indicated such transcripts only in early (ring) stages. We investigated this discrepancy by Northern blot, with probes covering a diverse var gene repertoire. We confirm that almost all var transcript types were detected only in ring stages. However, one type, the well-conserved varCSA transcript, was present constitutively in different laboratory parasites and does not appear to undergo antigenic variation. Although varCSA has been shown to encode a chondroitin sulphate A (CSA)-binding PfEMP1, we find that the presence of full-length varCSA transcripts does not correlate with the CSA-binding phenotype
Identification and characterization of human PCDH10 gene promoter
No full textRecent studies have suggested roles for PCDH10 as a novel tumor suppressor gene. In our previous work, we located the core promoter of PCDH10 to a 462-bp segment of 5'-flanking region characterized by a high GC content. Here we further identified and characterized the promoter for PCDH10. Transient transfection of PC3 and LNCaP cells with a series of deleted promoter constructs indicated that the minimal promoter region was between nucleotides - 144 and -99. This segment contained a CAAT box, a GT box, and a putative transcription factor binding site for AP-4. Mutational analysis identified that the CAAT box and GT box are necessary for promoter activity. Ectopic expression of NF-Ys increased reporter gene activity, whereas expression of a dominant-negative NF-YA decreased reporter gene activity. Co-transfection of Sp1/Sp3 expression plasmids enhanced reporter gene activity in a dose-dependent manner. Mithramycin A. an inhibitor of Sp-DNA interaction, reduced PCDH10 promoter activity. Electrophoretic mobility shift assays and chromatin immunoprecipitation demonstrated binding of transcription factors Sp1/Sp3 to the promoter region in vitro and in vivo. Our data show that Spl/Sp3 and CBF/NF-Y transcription factors play a crucial role in the basal expression of the human PCDH10 gene. (C) 2011 Elsevier B.V. All rights reserved.Genetics & HereditySCI(E)PubMed10ARTICLE149-5647
Novel deletion mutation of TRPS1 gene in a Chinese patient of trichorhinophalangeal syndrome type I
No full textTricho-rhino-phalangeal syndrome (TRPS) is a rare autosomal dominant disorder. Deletion or mutation of the TRPS1 gene leads to the tricho-rhino-phalangeal syndromes type I or type III. In this article, we describe a Chinese patient affected with type I TRPS and showing prominent pilar, rhinal and phalangeal abnormalities. Mutational screening and sequence analysis of TRPS1 gene revealed a previously unidentified four-base-pair deletion of nucleotides 1783-1786 (c.1783_1786delACTT). The mutation causes a frame shift after codon 593, introducing a premature stop codon after 637 residues in the gene sequence. This deletion is an unquestionable loss-of-function mutation, deleting all the functionally important parts of the protein. Our novel discovery indicates that sparse hair and metacarpal defects of tricho-rhino-phalangeal syndromes in this patient are due to this TRPS1 mutation. And this data further supports the critical role of TRPS1 gene in hair and partial skeleton morphogenesis. (C) 2013 Elsevier B.V. All rights reserved.Genetics & HereditySCI(E)PubMed0ARTICLE188-9152
The 5 '-upstream region of human programmed cell death 5 gene contains a highly active TATA-less promoter that is up-regulated by etoposide
No full textThe PDCD5 (programmed cell death 5), a novel apoptosis related gene, is functionally associated with cell apoptosis, exhibits a ubiquitous expression pattern and is up-regulated in some types of tumor cells undergoing apoptosis. To study the transcriptional regulation of the PDCD5 gene, we have cloned 1.1 kb of its 5'-upstream region. The DNA sequencing analysis revealed a major transcriptional start site at 72 base pairs in front of the ATG translational start codon. The upstream of the transcriptional start site lacks a canonical TATA box and CAAT box. Transient transfection and luciferase assay demonstrate that this region presents extremely strong promoter activity. The 5' deleted sequences fused to a luciferase reporter gene demonstrated that the - 555/ - 383 region from the transcription start site is crucial for transcriptional regulation, and the luciferase reporter gene's expression significantly increased in the early stage of cell apoptosis induced by etoposide. These results imply that the PDCD5 gene may be a target gene under the control of some important apoptosis-related transcriptional factors during the cell apoptosis. (C) 2004 Elsevier B.V All rights reserved.Genetics & HereditySCI(E)PubMed6ARTICLE39-4932
Sculpture by Gene Buckley
No full textAn art exhibit in Schaefer Fine Arts Center showing sculptures by Gene Buckle
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