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At least 1,045 records · Page 58Linked to original sources
PubMed2005

Rapid self-assembly of DNA on a microfluidic chip.

BACKGROUND: DNA self-assembly methods have played a major role in enabling methods for acquiring genetic information without having to resort to sequencing, a relatively slow and costly procedure. However, even self-assembly processes tend to be very slow when they rely upon diffusion on a large scale. Miniaturisation and integration therefore hold the promise of greatly increasing this speed of operation. RESULTS: We have developed a rapid method for implementing the self-assembly of DNA within a microfluidic system by electrically extracting the DNA from an environment containing an uncharged denaturant. By controlling the parameters of the electrophoretic extraction and subsequent analysis of the DNA we are able to control when the hybridisation occurs as well as the degree of hybridisation. By avoiding off-chip processing or long thermal treatments we are able to perform this hybridisation rapidly and can perform hybridisation, sizing, heteroduplex analysis and single-stranded conformation analysis within a matter of minutes. The rapidity of this analysis allows the sampling of transient effects that may improve the sensitivity of mutation detection. CONCLUSIONS: We believe that this method will aid the integration of self-assembly methods upon microfluidic chips. The speed of this analysis also appears to provide information upon the dynamics of the self-assembly process.

Journal Article↗
PubMed2000

Identification of a short cis-acting element in the human vasopressin type 2 receptor gene which confers high-level expression of a reporter gene specifically in collecting duct cells.

In the kidney, water reabsorption is mainly regulated by the binding of arginine vasopressin to vasopressin type 2 (V2) receptors. These receptors are expressed selectively in principal cells of the collecting ducts. To identify molecular mechanisms responsible for the cell-specific expression of the V2 receptor, we have analyzed the proximal promoter of the corresponding gene. We report the identification of a 33-bp enhancer [collecting duct tissue-specific element 1 (CSE1)] that induced high levels of expression of the luciferase reporter gene in three collecting duct cell lines, but not in other renal cell lines. In gel shift assays, CSE1 bound a DNA-binding protein expressed selectively in collecting duct cell lines, and a 7-bp mutation, which abolished the activity of CSE1 in transient transfection experiments, also abolished the binding of this protein. Furthermore, decoy experiments performed using CSE1 showed that this sequence was involved not only in the expression of a construct containing 4.2 kb of the V2 receptor proximal promoter, but also in the expression of the endogenous V2 receptor gene. CSE1 appears to act mostly by counteracting the inhibitory effects of a strong ubiquitous repressor element that we called CIE1. Collectively, these results identify the first functional collecting duct-specific cis-acting element.

3T3 Cells↗
PubMed1999

Characterization of the promoter for the human antisense fibroblast growth factor-2 gene; regulation by Ets in Jurkat T cells.

Human lymphoid cells were found to synthesize predominantly antisense, and not sense, fibroblast growth factor-2 (FGF-2) mRNA. Two cDNAs corresponding to human 1069- and 1173-nucleotide antisense FGF-2 mRNAs were cloned from Jurkat T cells. The two cDNAs each possess a unique exon 1 and common exon 2, 3, 4, and 5 sequences. Exon 4 and 5 sequences overlap in the 3' untranslated region of FGF-2 cDNA, but not in the FGF-2 open reading frame. This is unlike the Xenopus antisense FGF-2 homologue, which overlaps with parts of both the FGF-2 3' untranslated region and its open reading frame. To investigate the regulation of human antisense FGF-2 gene expression, a 2.5-kilobase (kb) promoter region was isolated and characterized. Transient transfection of promoter-luciferase constructs demonstrated the antisense FGF-2 promoter to be active in Jurkat cells. Using transient transfection and in vitro binding assays, specific mutations within the promoter sequence have implicated that Ets-like transcription factors are significant in regulating the human antisense FGF-2 gene in Jurkat cells.

Animals↗
PubMed2000

Histone H1 and H3 dephosphorylation are differentially regulated by radiation-induced signal transduction pathways.

We recently demonstrated that linker histone H1, which is thought to have a fundamental role in higher-order chromatin structure, becomes transiently dephosphorylated after ionizing radiation (IR) in a mutated ataxia telangiectasia (ATM) dependent manner. To establish whether H1 dephosphorylation was a component of a damage-response pathway that included dephosphorylation of other histones, we asked whether H3 was dephosphorylated in response to IR in a manner similar to H1. H1 and H3 are maximally phosphorylated in metaphase and both are dephosphorylated after IR. However, the duration of IR-induced H3 dephosphorylation is significantly longer than that of IR-induced H1 dephosphorylation. Moreover, H1 dephosphorylation is ATM-dependent, whereas H3 dephosphorylation is ATM-independent. These observations suggest that the damage-sensing pathways regulating H3 and H1 dephosphorylation diverge upstream of ATM.

Animals↗
PubMed2001

Inducible expression of the megakaryocyte-specific gene glycoprotein IX is mediated through an Ets binding site and involves upstream activation of extracellular signal-regulated kinase.

Glycoprotein IX is a megakaryocyte-specific gene crucial for adequate and functional expression of the Glycoprotein Ib-IX complex. This study used phorbol 12-myristate 13-acetate (PMA) and thrombopoietin (TPO)-induced differentiation of Dami and UT-7 cells, respectively, to investigate the regulation of inducible Glycoprotein IX expression during megakaryocyte differentiation. PMA and TPO were able to modulate GPIX expression at mRNA and protein levels. Transient transfection studies using nested 5'-deletions and mutations of the GPIX promoter demonstrated the absolute requirement of an inverted Ets site 5'-ACTTCCT-3' for inducible reporter gene expression. The upstream signaling events associated with PMA and TPO-inducible expression of GPIX were also investigated. The mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase inhibitor PD98059 inhibited both PMA and TPO-inducible reporter activity in a dose-dependent manner, whereas inhibition of p38/MAPK had no significant effect. The protein kinase C inhibitor GF109203X failed to inhibit TPO-activation of the GPIX promoter in UT-7 cells. This study demonstrates that inducible expression in response to either PMA or TPO is mediated through the Ets site in the proximal promoter of GPIX and is dependent upon the upstream activation of MAPK/extracellular signal-regulated kinase.

Amino Acid Motifs↗
PubMed2004

Development of cell cultures with competency for contributing to the zebrafish germ line.

The zebrafish is an established model for the genetic analysis of vertebrate development. Forward-genetic screens have generated thousands of mutations, and antisense-based methods have been used to transiently knockdown gene expression during embryogenesis. Although these methods have made the zebrafish a valuable system for the identification and functional characterization of developmentally important genes, one deficiency of the zebrafish model is the absence of methods to introduce targeted mutations to generate knockout lines of fish. Application of gene-targeting methods has been limited in nonmurine species due to the absence of germ-line competent embryonic stem (ES) cell lines. Recently, progress was made in addressing this problem by the derivation of zebrafish embryo cell lines that remain pluripotent and germ-line competent for multiple passages in culture. Zebrafish germ-line chimeras were generated using cultures derived from embryos at two different developmental stages, and targeted insertion of vector DNA by homologous recombination was demonstrated in both cultures. Several strategies are being used to optimize the production and identification of germ-line chimeras. The zebrafish embryo cell culture system should provide the basis of a gene-targeting approach that will complement other genetic strategies and improve the utility of the zebrafish model for studies of development and disease.

Animals↗
PubMed1991

Regulatory elements required for light-mediated expression of the Petroselinum crispum chalcone synthase gene.

Chalcone synthase (CHS) catalyzes the committed enzymatic step in flavonoid biosynthesis. In parsley (Petroselinum crispum), CHS is encoded by a single gene locus. Transcriptional activation of the gene in response to UV-containing white light has been demonstrated. Analysis of the CHS gene promoter by in vivo footprinting revealed four short sequences, designated Boxes I, II, III, and IV, which contain guanosine residues with altered reactivity to the methylating agent dimethylsulfate in UV-treated versus untreated parsley cells. Studies were performed to characterize the functional components of the CHS gene promoter using a parsley protoplast transient expression system. By deletion and block-mutation analyses it was shown that Boxes I and II act together as a cis-acting unit and are necessary components of the minimal, light-responsive CHS gene promoter. The Box II sequence, which is similar to the conserved G Box sequence defined in promoters of ribulose 1,5-bisphosphate carboxylase small subunit (RBCS) genes, has been subjected to detailed analysis by site-directed mutagenesis. The heptameric sequence 5'-ACGTGGC-3' has been defined as the critical core of Box II required for light induction in the context of the CHS gene minimal promoter. Box II is functionally equivalent to a second, sequence-related element (Box III) that can replace Box II in an orientation-dependent manner. Chimaeric promoter-fusion constructs to the GUS reporter gene demonstrated that Boxes I and II, together constituting a cis-acting unit, are necessary and sufficient for light-mediated activation of the CHS gene promoter.

Acyltransferases↗
PubMed1994

[Spread of mutations in an inhomogeneous environment with respect to migration. III. A closed model of population dynamics with a arbitrary type of mutation and analysis of the effect of selection mechanisms and "death-reproduction"].

A closed mathematical model of population dynamics for an arbitrary mutant type was developed based on results obtained previously. The model considers the number of individuals with different genotypes, their random and constrained movements across the range, mechanisms of selection and Death-reproduction, and different conditions of the process at range boundaries. The influence of mechanisms of selection and Death-reproduction on integral parameters of the studied process was analyzed.

Death↗
PubMed1994

Biochemical mechanism of HIV-I Vpr function. Specific interaction with a cellular protein.

vpr is an accessory gene of human immunodeficiency virus I (HIV-I). Although unnecessary for viral replication in T cell lines, growing evidence suggests that it is essential for virus replication in monocytes/macrophages and for replication in vivo. We expressed HIV-I vpr in Escherichia coli and purified Vpr by affinity chromatography. In a coprecipitation assay, the purified Vpr interacted specifically with a cellular protein designated as Vpr-interacting protein, or RIP. Mutational analysis suggested that this interaction required a domain rich in leucine/isoleucine residues and highly conserved between HIV-I and SIVmac Vprs. During transient expression in mammalian cells, HIV-I Vpr was localized in the nucleus. However, mutational analysis failed to identify in Vpr a typical nuclear localization signal rich in basic amino acid residues. Instead, Vpr nuclear localization seemed to correlate with Vpr interaction with RIP. Mutations in the C-terminal 20-amino acid region containing a cryptic nuclear localization signal did not abolish Vpr nuclear localization or interaction with RIP, whereas point mutations in the leucine/isoleucine-rich domain abolished Vpr interaction with RIP and rendered Vpr unstable during transient expression. These results suggest that RIP may be involved in Vpr function.

Amino Acid Sequence↗
PubMed2005

Mutations affecting starch synthase III in Arabidopsis alter leaf starch structure and increase the rate of starch synthesis.

The role of starch synthase (SS) III (SSIII) in the synthesis of transient starch in Arabidopsis (Arabidopsis thaliana) was investigated by characterizing the effects of two insertion mutations at the AtSS3 gene locus. Both mutations, termed Atss3-1 and Atss3-2, condition complete loss of SSIII activity and prevent normal gene expression at both the mRNA and protein levels. The mutations cause a starch excess phenotype in leaves during the light period of the growth cycle due to an apparent increase in the rate of starch synthesis. In addition, both mutations alter the physical structure of leaf starch. Significant increases were noted in the mutants in the frequency of linear chains in amylopectin with a degree of polymerization greater than approximately 60, and relatively small changes were observed in chains of degree of polymerization 4 to 50. Furthermore, starch in the Atss3-1 and Atss3-2 mutants has a higher phosphate content, approximately two times that of wild-type leaf starch. Total SS activity is increased in both Atss3 mutants and a specific SS activity appears to be up-regulated. The data indicate that, in addition to its expected direct role in starch assembly, SSIII also has a negative regulatory function in the biosynthesis of transient starch in Arabidopsis.

Arabidopsis↗
PubMedDate not supplied

[Catabolyte repression of Escherichia coli K12 mutants with defects in different systems of glucose transport].

The phenomenon of glucose catabolite repression was studied in E. coli mutants inable to transport this carbohydrate. The pts 1, H mutant P34 was much less sensitive to the repressive effect of glucose on beta-galactosidase synthesis than the parent type. The 1103 mutant devoid of enzyme 1 of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) behaves in the same way as P34 mutant after addition of glucose to casamino acid mineral medium. However, in minimal medium with succinate as the sole source of carbon, cells of the 1103 mutant show enhanced sensibility to transient glucose repression. The effect of hypersensibility disappears when the lac I mutation leading to constitutive the beta-galactosidase synthesis is introduced in 1103 mutant. It is shown that the enhanced sensibility of beta-galactosidase synthesis to glucose transient repression in 1103 mutant is an effect of the aburpt decrease in its growth rate in the presence of succinate and most probably this decrease leads to "inducer exclusion" of the lac operon. It is also shown that if one introduces the P34 mutation in strain JD3 devoid of one of the enzymes II for glucose (and due to this resistant to glucose catabolite respression) then the level of resistance in double mutant does not increase in spite of considerable supression of 14C glucose accumulation. In connection with this the role is discussed of separate components of the E. coli K 12 glucose transport system in realization of the phenomenon of catabolite repression.

Biological Transport, Active↗
PubMed2001

A unique downstream estrogen responsive unit mediates estrogen induction of proteinase inhibitor-9, a cellular inhibitor of IL-1beta- converting enzyme (caspase 1).

Recently, proteinase inhibitor 9 (PI-9) was identified as the first endogenous inhibitor of caspase 1 (IL-1beta-converting enzyme). The regulation of PI-9 expression, therefore, has great importance in the control of inflammatory processes. We reported that PI-9 mRNA and protein are rapidly and directly induced by estrogen in human liver cells. Using transient transfections to assay PI-9 promoter truncations and mutations, we demonstrate that this strong estrogen induction is mediated by a unique downstream estrogen responsive unit (ERU) approximately 200 nucleotides downstream of the transcription start site. Using primers flanking the ERU in chromatin immunoprecipitation assays, we demonstrate estrogen-dependent binding of ER to the cellular PI-9 promoter. The ERU consists of an imperfect estrogen response element (ERE) palindrome immediately adjacent to a direct repeat containing two consensus ERE half-sites separated by 13 nucleotides (DR13). In transient transfections, all four of the ERE half-sites in the imperfect ERE and in the DR13 were important for estrogen inducibility. Transfected chicken ovalbumin upstream transcription factor I and II down-regulated estrogen-mediated expression from the ERU. EMSAs using purified recombinant human ERalpha demonstrate high-affinity binding of two ER complexes to the ERU. Further EMSAs showed that one ER dimer binds to an isolated DR13, supporting the view that one ER dimer binds to the imperfect ERE and one ER dimer binds to DR13. Deoxyribonuclease I footprinting showed that purified ER protected all four of the half-sites in the ERU. Our finding that a direct repeat can function with an imperfect ERE palindrome to confer estrogen inducibility on a native gene extends the repertoire of DNA sequences able to function as EREs.

COUP Transcription Factor I↗
PubMed2002

An osmotically induced cytosolic Ca2+ transient activates calcineurin signaling to mediate ion homeostasis and salt tolerance of Saccharomyces cerevisiae.

Hyperosmotic stress caused by NaCl, LiCl, or sorbitol induces an immediate and short duration ( approximately 1 min) transient cytosolic Ca(2+) ([Ca(2+)](cyt)) increase (Ca(2+)-dependent aequorin luminescence) in Saccharomyces cerevisiae cells. The amplitude of the osmotically induced [Ca(2+)](cyt) transient was attenuated by the addition of chelating agents EGTA or BAPTA, cation channel pore blockers, competitive inhibitors of Ca(2+) transport, or mutations (cch1Delta or mid1Delta) that reduce Ca(2+) influx, indicating that Ca(ext)(2+) is a source for the transient. An osmotic pretreatment (30 min) administered by inoculating cells into media supplemented with either NaCl (0.4 or 0.5 m) or sorbitol (0.8 or 1.0 m) enhanced the subsequent growth of these cells in media containing 1 m NaCl or 2 m sorbitol. Inclusion of EGTA in the osmotic pretreatment media or the cch1Delta mutation reduced cellular capacity for NaCl but not hyperosmotic adaptation. The stress-adaptive effect of hyperosmotic pretreatment was mimicked by exposing cells briefly to 20 mm CaCl(2). Thus, NaCl- or sorbitol-induced hyperosmotic shock causes a [Ca(2+)](cyt) transient that is facilitated by Ca(2+) influx, which enhances ionic but not osmotic stress adaptation. NaCl-induced ENA1 expression was inhibited by EGTA, cch1Delta mutation, and FK506, indicating that the [Ca(2+)](cyt) transient activates calcineurin signaling to mediate ion homeostasis and salt tolerance.

Calcineurin↗
PubMed2001

Analysis of the phosphorylation status of Epstein-Barr virus LMP2A in epithelial cells.

LMP2A deletion and point mutants, with mutations in phosphotyrosine-containing protein-protein interaction motifs, were transiently expressed in 293 cells and their phosphorylation was examined in immune complex kinase assays as well as in vivo. In vitro LMP2A phosphorylation depended on tyrosine 112. In vivo, mutations of single tyrosines did not eliminate LMP2 phosphorylation, although mutation of the LMP2A ITAM decreased LMP2A phosphorylation. The relationship between LMP2A in vitro phosphorylation and that induced by cell-extracellular matrix (ECM) interactions was also investigated. While LMP2A was phosphorylated to higher levels in whole-cell extracts of stimulated cells, a difference in in vitro kinase assays with extracts from stimulated and unstimulated cells was not detected, indicating that the ECM-mediated regulation of LMP2A phosphorylation is lost in vitro. In the presence of LMP2A, several cellular proteins with molecular weights between 70 and 80 kDa were phosphorylated on tyrosine. This increase in cellular protein phosphorylation depended on the LMP2A ITAM motif and suggests that the ITAM may participate in signal-transduction events in epithelial cells.

Binding Sites↗
PubMed2005

Transmission and accumulation of CTL escape variants drive negative associations between HIV polymorphisms and HLA.

Human immunodeficiency virus (HIV)-1 amino acid sequence polymorphisms associated with expression of specific human histocompatibility leukocyte antigen (HLA) class I alleles suggest sites of cytotoxic T lymphocyte (CTL)-mediated selection pressure and immune escape. The associations most frequently observed are between expression of an HLA class I molecule and variation from the consensus sequence. However, a substantial number of sites have been identified in which particular HLA class I allele expression is associated with preservation of the consensus sequence. The mechanism behind this is so far unexplained. The current studies, focusing on two examples of "negatively associated" or apparently preserved epitopes, suggest an explanation for this phenomenon: negative associations can arise as a result of positive selection of an escape mutation, which is stable on transmission and therefore accumulates in the population to the point at which it defines the consensus sequence. Such negative associations may only be in evidence transiently, because the statistical power to detect them diminishes as the mutations accumulate. If an escape variant reaches fixation in the population, the epitope will be lost as a potential target to the immune system. These data help to explain how HIV is evolving at a population level. Understanding the direction of HIV evolution has important implications for vaccine development.

AIDS Vaccines↗
PubMed1992

A stem cell niche theory of intestinal crypt maintenance based on a study of somatic mutation in colonic mucosa.

In both large and small intestine, mutagen administration leads to the occurrence of isolated crypts that are completely populated by a mutated phenotype; therefore, it has been proposed that crypts are maintained by a single stem cell. We show in mice that a single dose of mutagen leads to an early transient increase in frequency of colonic crypts that show a partial mutated phenotype and a later increase in frequency of crypts that show a complete mutated phenotype. This increase reaches a plateau at about the same time as the disappearance of partially mutated crypts. The same is true in the small intestine, but the time course is much slower. We propose an explanation based on multiple crypt stem cells that occupy a "stem cell niche," with random cell loss after stem cell division. A small difference in the number of crypt stem cells that occupy the niche provides a simple explanation for the surprisingly large difference in the time course of phenotypic changes in the large and small intestines after administration of a single dose of mutagen.

Animals↗
PubMed1997

Glucocorticoids use a positive liver element to repress fibrate-induced adipose transcription of the phosphoenolpyruvate carboxykinase gene.

Glucocorticoids inhibit basal and hormone-induced phosphoenolpyruvate carboxykinase (PEPCK) gene transcription in adipocytes whereas beta-adrenergic agonists and fibrates are stimulatory. Here we show that dexamethasone inhibits the induction of PEPCK mRNA by isoprenaline or clofibrate in 3T3-F442A adipocytes. RU 38486 antagonizes dexamethasone effect, suggesting the involvement of the glucocorticoid receptor. In H4IIE hepatoma cells, glucocorticoids enhance PEPCK gene transcription through a complex region which encompasses an element, AF1, with a direct repeat 1-type sequence. Mutations in the AF1 sequence abolish binding of nuclear factors from liver and from 3T3-F442A adipocytes. We transiently transfected 3T3-F442A cells with a wild type or an AF1-mutated PEPCK-CAT construct comprising -2100 to +69 base pairs of the promoter fused to the chloramphenicol acetyltransferase (CAT) gene. With both constructs, CAT activity is decreased by dexamethasone and is increased by isoprenaline or by clofibrate. However, dexamethasone is unable to inhibit clofibrate induction of CAT activity in cells transfected with the AF1-mutated construct whereas it prevents isoprenaline action on both constructs. Hence, although a single hormone can repress stimulations originating from different intracellular routes, sites in the promoter which mediate inhibition of a specific stimulation are distinct.

3T3 Cells↗
PubMed1995

Expression studies of two vasopressin V2 receptor gene mutations, R202C and 804insG, in nephrogenic diabetes insipidus.

Nephrogenic diabetes insipidus (NDI) is a rare X-linked disorder associated with renal tubule resistance to arginine vasopressin (AVP). To understand the mechanisms of AVP resistance underlying this disorder, we have analyzed the vasopressin V2 receptor gene in two unrelated Japanese kindreds with NDI and expressed the mutants to characterize their functional properties. Direct sequencing revealed two V2 receptor gene mutations: a missense mutation from Arg202 to Cys in the third extracellular domain (R202C) and a single base insertion (G) in two consecutive GGG triplets (nucleotide 804 to 809) in the third cytoplasmic domain, resulting in a frame shift with premature termination at codon 258 (804insG). Transient expression study with COS-7 cells showed that R202C mutation reduced both binding affinity (15%) and capacity (30%), while 804insG mutation abolished binding ability. For further evaluation of the binding ability of the R202C mutant, we expressed the mutants in Chinese hamster ovary (CHO) cells. Although the mutant cell lines produced V2 receptor mRNA comparable levels to the wild-type receptor cell lines, R202C mutant cell lines had no binding ability. Our results suggest an introduction of a new cysteine residue in the extracellular domain and a receptor truncation removing one third of the carboxyl terminus could impair ligand binding activity of the V2 receptor through a post-transcriptional mechanism, thereby causing AVP resistance in the NDI patients.

Adult↗