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At least 1,171 records · Page 65Linked to original sources
PubMed2004

A library of siRNA duplexes targeting the phosphoinositide 3-kinase pathway: determinants of gene silencing for use in cell-based screens.

Gene silencing through RNA interference (RNAi) has been established as a means of conducting reverse genetic studies. In order to better understand the determinants of short interfering RNA (siRNA) knockdown for use in high-throughput cell-based screens, 148 siRNA duplexes targeting 30 genes within the PI3K pathway were selected and synthesized. The extent of RNA knockdown was measured for 22 genes by quantitative real-time PCR. Analysis of the parameters correlating with effective knockdown showed that (i) duplexes targeting the middle of the coding sequence silenced significantly poorer, (ii) silencing by duplexes targeting the 3'UTR was comparable with duplexes targeting the coding sequence, (iii) pooling of four or five duplexes per gene was remarkably efficient in knocking down gene expression and (iv) among duplexes that achieved a >70% knockdown of the mRNA there were strong nucleotide preferences at specific positions, most notably positions 11 (G or C) and 19 (T) of the siRNA duplex. Finally, in a proof-of-principle pathway-wide cell-based genetic screen, conducted to detect negative genetic regulators of Akt S473 phosphorylation, both known negative regulators of this phosphorylation, PTEN and PDK1, were found. These data help to lay the foundation for genome-wide siRNA screens in mammalian cells.

Cell Line↗
PubMed2004

2'-deoxyribonolactone lesion produces G->A transitions in Escherichia coli.

2'-deoxyribonolactone (dL) is a C1'-oxidized abasic site damage generated by a radical attack on DNA. Numerous genotoxic agents have been shown to produce dL including UV and gamma-irradiation, ene-dye antibiotics etc. At present the biological consequences of dL present in DNA have been poorly documented, mainly due to the lack of method for introducing the lesion in oligonucleotides. We have recently designed a synthesis of dL which allowed investigation of the mutagenicity of dL in Escherichia coli by using a genetic reversion assay. The lesion was site-specifically incorporated in a double-stranded bacteriophage vector M13G*1, which detects single-base-pair substitutions at position 141 of the lacZalpha gene by a change in plaque color. In E.coli JM105 the dL-induced reversion frequency was 4.7 x 10(-5), similar to that of the classic abasic site 2'-deoxyribose (dR). Here we report that a dL residue in a duplex DNA codes mainly for thymidine. The processing of dL in vivo was investigated by measuring lesion-induced mutation frequencies in DNA repair deficient E.coli strains. We showed a 32-fold increase in dL-induced reversion rate in AP endonuclease deficient (xth nfo) mutant compared with wild-type strain, indicating that the Xth and Nfo AP endonucleases participate in dL repair in vivo.

Bacteriophage M13↗
PubMed2006

A semi-automated high-throughput approach to the generation of transposon insertion mutants in the nematode Caenorhabditis elegans.

The generation of a large collection of defined transposon insertion mutants is of general interest to the Caenorhabditis elegans research community and has been supported by the European Union. We describe here a semi-automated high-throughput method for mutant production and screening, using the heterologous transposon Mos1. The procedure allows routine culture of several thousand independent nematode strains in parallel for multiple generations before stereotyped molecular analyses. Using this method, we have already generated >17 500 individual strains carrying Mos1 insertions. It could be easily adapted to forward and reverse genetic screens and may influence researchers faced with making a choice of model organism.

Animals↗
PubMed2006

Sequence-dependent enhancement of hydrolytic deamination of cytosines in DNA by the restriction enzyme PspGI.

Hydrolytic deamination of cytosines in DNA creates uracil and, if unrepaired, these lesions result in C to T mutations. We have suggested previously that a possible way in which cells may prevent or reduce this chemical reaction is through the binding of proteins to DNA. We use a genetic reversion assay to show that a restriction enzyme, PspGI, protects cytosines within its cognate site, 5'-CCWGG (W is A or T), against deamination under conditions where no DNA cleavage can occur. It decreases the rate of cytosine deamination to uracil by 7-fold. However, the same protein dramatically increases the rate of deaminations within the site 5'-CCSGG (S is C or G) by approximately 15-fold. Furthermore, a similar increase in cytosine deaminations is also seen with a catalytically inactive mutant of the enzyme showing that endonucleolytic ability of the protein is dispensable for its mutagenic action. The sequences of the mutants generated in the presence of PspGI show that only one of the cytosines in CCSGG is predominantly converted to thymine. Our results are consistent with PspGI 'sensitizing' the cytosine in the central base pair in CCSGG for deamination. Remarkably, PspGI sensitizes this base for damage despite its inability to form stable complexes at CCSGG sites. These results can be explained if the enzyme has a transient interaction with this sequence during which it flips the central cytosine out of the helix. This prediction was validated by modeling the structure of PspGI-DNA complex based on the structure of the related enzyme Ecl18kI which is known to cause base-flipping.

Base Sequence↗
PubMed2002

Efficient isolation of targeted Caenorhabditis elegans deletion strains using highly thermostable restriction endonucleases and PCR.

Reverse genetic approaches to understanding gene function would be greatly facilitated by increasing the efficiency of methods for isolating mutants without the reliance on a predicted phenotype. Established PCR-based methods of isolating deletion mutants are widely used for this purpose in Caenorhabditis elegans. However, these methods are inefficient at isolating small deletions. We report here a novel modification of PCR-based methods, employing thermostable restriction enzymes to block the synthesis of wild-type PCR product, so that only the deletion PCR product is amplified. This modification greatly increases the efficiency of isolating small targeted deletions in C.elegans. Using this method six new deletion strains were isolated from a small screen of approximately 400 000 haploid genomes, most with deletions <1.0 kb. Greater PCR detection sensitivity by this modification permitted approximately 10-fold greater pooling of DNA samples, reducing the effort and reagents required for screens. In addition, effective suppression of non-specific amplification allowed multiplexing with several independent primer pairs. The increased efficiency of this technique makes it more practical for small laboratories to undertake gene knock-out screens.

Animals↗
PubMed2003

Establishment of conditional vectors for hairpin siRNA knockdowns.

Small interference RNA (siRNA) is an emerging methodology in reverse genetics. Here we report the development of a new tetracycline-inducible vector-based siRNA system, which uses a tetracycline-responsive derivative of the U6 promoter and the tetracycline repressor for conditional in vivo transcription of short hairpin RNA. This method prevents potential lethality immediately after transfection of a vector when the targeted gene is indispensable, or the phenotype of the knockdown is lethal or results in a growth abnormality. We show that the controlled knockdown of DNA methyltransferase 1 (DNMT1) in human cancer resulted in growth arrest. Removal of the inducer, doxycycline, from treated cells led to re-expression of the targeted gene. Thus the method allows for a highly controlled approach to gene knockdown.

Base Sequence↗
PubMed2003

Stable and heritable gene silencing in the malaria vector Anopheles stephensi.

Heritable RNA interference (RNAi), triggered from stably expressed transgenes with an inverted repeat (IR) configuration, is an important tool for reverse genetic studies. Here we report on the development of stable RNAi in Anopheles stephensi mosquitoes, the major vector of human malaria in Asia. Trans genic mosquitoes stably expressing a RNAi transgene, designed to produce intron-spliced double-stranded RNA (dsRNA) targeting the green fluorescent protein EGFP gene, were crossed to an EGFP-expressing target line. EGFP expression was dramatically reduced at both the protein and RNA levels. The levels of gene silencing depended upon the RNAi gene copy number and its site of integration. These results demonstrate that specific RNAi-mediated knockdown of gene function can be achieved with high efficiency in Anopheles . This will be invaluable to systematically unravel the function of Anopheles genes determining the vectorial capacity of the malaria parasite.

Animals↗
PubMed2004

Spatially and temporally controlled gene transfer by electroporation into adherent cells on plasmid DNA-loaded electrodes.

Functional characterization of human genes is one of the most challenging tasks in current genomics. Owing to a large number of newly discovered genes, high-throughput methodologies are greatly needed to express in parallel each gene in living cells. To develop a method that allows efficient transfection of plasmids into adherent cells in spatial- and temporal-specific manners, we studied electric pulse-triggered gene transfer using a plasmid-loaded electrode. A plasmid was loaded on a gold electrode surface having an adsorbed layer of poly(ethyleneimine), and cells were then plated directly onto this modified surface. The plasmid was detached from the electrode by applying a short electric pulse and introduced into the cells cultured on the electrode, resulting in efficient gene expression, even in primary cultured cells. The location of transfected cells could be restricted within a small area on a micropatterned electrode, showing the versatility of the method for spatially controlled transfection. Plasmid transfection could also be performed in a temporally controlled manner without a marked loss of the efficiency when an electric pulse was applied within 3 days after cell plating. The method described here will provide an efficient means to transfer multiple genes, in parallel, into cultured mammalian cells for high-throughput reverse genetics research.

Animals↗
PubMed2005

A bi-functional siRNA construct induces RNA interference and also primes PCR amplification for its own quantification.

RNA interference (RNAi) is a process of post-transcriptional gene silencing initiated by double-stranded RNAs, including short interfering RNA (siRNA). Silencing is sequence-specific and RNAi has rapidly become central to the study of gene function. RNAi also carries promise for selective silencing of viral and endogenous genes causal for disease. To detect the very low levels of siRNA effective for RNAi we modified the 3' end of the sense strand of siRNA with a nuclease-resistant DNA hairpin. We show that the modified siRNA-DNA construct (termed 'crook' siRNA) functions as a primer for the PCR and describe a novel, yet simple PCR protocol for its quantification (amolar levels/cell). When transfected into mammalian cells, crook siRNA induces selective mRNA knock-down equivalent to its unmodified siRNA counterpart. This new bifunctional siRNA construct will enable future in vivo studies on the uptake, distribution and pharmacokinetics of siRNA, and is particularly important for the development of siRNA-based therapeutics. More generally, PCR-based detection of siRNA carries wide-ranging applications for RNAi reverse genetics.

Cell Line↗
PubMed2006

Ribonucleome analysis identified enzyme genes responsible for wybutosine synthesis.

Wybutosine (yW) is a hyper modified guanosine with a tricyclic base found at the 3'-position adjacent to the anticodon of eukaryotic phenylalanine tRNA. yW supports reading frame maintenance by stabilizing codon-anticodon interactions during decoding on the ribosome. To identify genes responsible for yW synthesis from uncharacterized genes of Saccharomyces cerevisiae, we employed a systematic reverse genetic approach combined with mass spectrometry ('ribonucleome analysis'). We found four new genes YPL207w, YML005w, YOL141w and YGL050w (named TYW1, TYW2, TYW3 and TYW4, respectively) to be responsible for wybutosine (yW) synthesis. Mass spectrometric analysis of each modification intermediate of yW revealed its sequential biosynthetic pathway. Multi-step enzymatic formation of yW was partially reconstituted in vitro using recombinant proteins. The results suggested that yW synthesis might proceed through sequential reactions in a complex formed by multiple protein components assembled with the precursor tRNA.

Gene Deletion↗
PubMed2005

Application of RNA interference to root-knot nematode genes encoding esophageal gland proteins.

Plant parasitic nematodes have been, so far, refractory to transformation or mutagenesis. The functional analysis of nematode genes relies on the development of reverse genetic tools adapted to these obligate parasites. Here, we describe the application of RNA interference (RNAi) to the root-knot nematode Meloidogyne incognita for the knock-down of two genes expressed in the subventral esophageal glands of the nematode and potentially involved in parasitism, the calreticulin (Mi-crt) and the polygalacturonase (Mi-pg-1) genes. Incubation in 1% resorcinol for 4 h induced double-stranded RNA uptake through the alimentary track of the nematodes and led to up to 92% depletion of Mi-crt transcripts. Timecourse analysis of the silencing showed different temporal patterns for Mi-crt and Mi-pg-1. The silencing of Mi-crt was optimal 20 h after soaking, whereas the silencing of Mi-pg-1 was optimal 44 h after soaking. For the two genes, the silencing effect was highly time-limited, since no transcript depletion was detectable 68 h after soaking.

Animals↗
PubMed2006

Targeted gene mutation in Phytophthora spp.

The genus Phytophthora belongs to the oomycetes and is composed of plant pathogens. Currently, there are no strategies to mutate specific genes for members of this genus. Whole genome sequences are available or being prepared for Phytophthora sojae, P. ramorum, P. infestans, and P. capsici and the development of molecular biological techniques for functional genomics is encouraged. This article describes the adaptation of the reverse-genetic strategy of targeting induced local lesions in genomes (TILLING) to isolate gene-specific mutants in Phytophthora spp. A genomic library of 2,400 ethylnitrosourea (ENU) mutants of P. sojae was created and screened for induced point mutations in the genes encoding a necrosisinducing protein (PsojNIP) and a Phytophthora-specific phospholipase D (PsPXTM-PLD). Mutations were detected in single individuals and included silent, missense, and nonsense changes. Homozygous mutant isolates carrying a potentially deleterious missense mutation in PsojNIP and a premature stop codon in PsPXTM-PLD were identified. No phenotypic effect has yet been found for the homozygous mutant of PsojNIP. For those of PsPXTM-PLD, a reduction in growth rate and an appressed mycelial growth was observed. This demonstrates the feasibility of target-selected gene disruption for Phytophthora spp. and adds an important tool for functional genomic investigation.

Algal Proteins↗
PubMed1999

Iron regulation and pathogenicity in Erwinia chrysanthemi 3937: role of the Fur repressor protein.

Low iron availability is a triggering signal for coordinated expression of the genes encoding pectate lyases PelB, PelC, PelD, and PelE, and chrysobactin iron transport functions, which are two main determinants of phytopathogenicity of the Erwinia chrysanthemi strain 3937. The possible implication of the ferric uptake regulation (Fur) protein in this process was investigated. The E. chrysanthemi fur gene was cloned by functional complementation of an Escherichia coli fur mutant and sequenced. The 444-bp open reading frame identified was found to code for a protein highly similar to the E. coli Fur regulator. An E. chrysanthemi fur null mutant was constructed by reverse genetics. This mutant showed altered growth capacity and reduced pathogenicity on African violets. In a fur background, transcriptional lacZ fusions to genes belonging to the E. chrysanthemi high affinity iron transport systems were constitutively expressed. Transcription of the pelA, pelD, and pelE genes was analyzed, using fusions to the uidA reporter gene. Iron availability and a fur mutation did not influence the expression of pelA. In the presence of iron, pelD and pelE transcription levels were higher in the fur mutant than in the parental strain. Furthermore, iron deficiency stimulated the expression of both fusions in the fur mutant. These findings indicate that, in E. chrysanthemi 3937, (i) Fur negatively controls iron transport and genes encoding PelD and PelE, and (ii) additional factor(s) mediate iron regulation of the pel genes.

Amino Acid Sequence↗
PubMed2001

Role of the nucleoid-associated protein H-NS in the synthesis of virulence factors in the phytopathogenic bacterium Erwinia chrysanthemi.

The ability of the enterobacterium Erwinia chrysanthemi to induce pathogenesis in plant tissue is strongly related to the massive production of plant-cell-wall-degrading enzymes (pectinases, cellulases, and proteases). Additional factors, including flagellar proteins and exopolysaccharides (EPS), also are required for the efficient colonization of plants. Production of these virulence factors, particularly pectate lyases, the main virulence determinant, is tightly regulated by environmental conditions. The possible involvement of the protein H-NS in this process was investigated. The E. chrysanthemi hns gene was cloned by complementation of an Escherichia coli hns mutation. Its nucleotide sequence contains a 405-bp open reading frame that codes for a protein with 85% identity to the E. coli H-NS protein. An E. chrysanthemi hns mutant was constructed by reverse genetics. This mutant displays a reduced growth rate and motility but an increased EPS synthesis and sensitivity toward high osmolarity. Furthermore, pectate lyase production is dramatically reduced in this mutant. The hns mutation acts on at least two conditions affecting pectate lyase synthesis: induction of pectate lyase synthesis at low temperatures (25 degrees C) is no longer observed in the hns mutant and induction of pectate lyase production occurs in the late stationary growth phase in the hns background, instead of in the late exponential growth phase as it does in the parental strain. Moreover, the E. chrysanthemi hns mutant displays reduced virulence on plants. Taken together, these data suggest that H-NS plays a crucial role in the expression of the virulence genes and in the pathogenicity of E. chrysanthemi.

Bacterial Proteins↗
PubMed2001

Essential role of superoxide dismutase on the pathogenicity of Erwinia chrysanthemi strain 3937.

The sodA gene from Erwinia chrysanthemi strain 3937 was cloned by functional complementation of an Escherichia coli sodA sodB mutant and sequenced. We identified a 639-bp open reading frame, which encodes a protein that is 85% identical to the E. coli manganese-containing superoxide dismutase MnSOD. Promoter elements of this gene were identified by transcriptional mapping experiments. We constructed an E. chrysanthemi deltasodA mutant by reverse genetics. The deltasodA mutation resulted in the absence of a cytoplasmic SOD, which displays the same characteristics as those of MnSOD. The deltasodA mutant was more sensitive to paraquat than the wild-type strain. This mutant could macerate potato tubers, similar to the wild-type strain. In contrast, when inoculated on African violets, the mutant produced, at most, only small necrotic lesions. If the inoculum was supplemented with the superoxide anion-scavenging metalloporphyrin MnTMPyP or purified SOD and catalase, the deltasodA mutant was able to macerate the inoculated zone. Generation of superoxide anion by African violet leaves inoculated with E. chrysanthemi was demonstrated with nitroblue tetrazolium as an indicator. Therefore, at the onset of infection, E. chrysanthemi cells encounter an oxidative environment and require active protective systems against oxidative damages such as MnSOD to overcome these types of conditions.

Amino Acid Sequence↗
PubMed2003

Multiple resistance phenotypes to Lettuce mosaic virus among Arabidopsis thaliana accessions.

With the aim to characterize plant and viral factors involved in the molecular interactions between plants and potyviruses, a Lettuce mosaic virus (LMV)-Arabidopsis thaliana pathosystem was developed. Screening of Arabidopsis accessions with LMV isolates indicated the existence of a large variability in the outcome of the interaction, allowing the classification of Arabidopsis accessions into seven susceptibility groups. Using a reverse genetic approach, the genome-linked protein of LMV, a multifunctional protein shown to be involved in viral genome amplification and movement of potyviruses, was established as the viral determinant responsible for the ability to overcome the resistance of the Niederzenz accession to LMV-0. Preliminary genetic analyses from F2 and recombinant inbred lines available between susceptible and resistant Arabidopsis accessions revealed the existence of at least three resistance phenotypes to LMV with different genetic bases. One dominant resistance gene, designated LLM1, involved in blocking the replication or cell-to-cell movement of the LMV-0 isolate in the Columbia accession, was mapped to chromosome I and shown to be linked to the marker nga280. At the same time, genetic analyses of segregating F2 populations were consistent with the restriction of the systemic movement of the LMV-AF199 isolate in Columbia being controlled by two dominant genes and with the complete resistance to all tested LMV isolates of the Cape Verde islands (Cvi) accession being conferred by a single recessive resistance gene. Sequencing of the eukaryotic translation initiation factor 4E genes from the different LMV-resistant Arabidopsis accessions showed that these factors are not directly involved in the characterized resistance phenotypes.

Amino Acid Sequence↗
PubMed1994

Stem cells and transcription factors in the development of the mammalian neural crest.

The neural crest is a migratory population of multipotent embryonic cells that generates the neurons and glia of the peripheral nervous system, as well as a variety of non-neural mesectodermal and endocrine cell types. The study of neural crest cell and molecular biology provides a system to investigate how such multipotent cells choose their fates, and whether the repertoire of fates becomes progressively restricted with time. The study of mammalian neural crest development has lagged behind studies of avian crest development due to the relative inaccessibility of mammalian embryos. The development of reverse genetic methods in mice, however, has made the analysis of mammalian neural crest development both more attractive and more tractable. Rodent neural crest cells have been isolated and grown in clonogenic cultures, where they behave as multipotent stem cells. This system provides an assay for factors that influence the differentiation of these multipotent cells. Transcription factors provide valuable early markers for neural crest cells as well as molecular handles on the lineage segregation process. One such factor is Mash1, a homolog of the Drosophila proneural genes, achaete-scute. Mash1 marks autonomic progenitor cells and is essential for their development in vivo, as shown by gene knockout experiments.

Animals↗
PubMed2006

A gene-targeting approach for functional characterization of KIAA genes encoding extremely large proteins.

Given that thousands of genes exist in the mammalian genome, criteria are needed to prioritize their functional analysis and to decrease the likelihood of producing gene-targeted mice that lack overt phenotypes. Initial analysis efforts are likely to be fruitful if focused on genes encoding large proteins, since at least some large proteins serve as frameworks for intricate assembly of protein complexes, and their inactivation would render definitive, observable phenotypes. Here, we describe the functional characterization of the murine homologues of five human KIAA genes (KIAA1409, KIAA1440, KIAA1447, KIAA1768, and KIAA1276) that encode large proteins. Gene-targeted mice had phenotypic and developmental defects resulting from the functional deletion of three of these five genes. Mice with targeted disruption of KIAA1409 lacked the ability to drink, and those with targeted disruption of KIAA1447 displayed hind leg motor dysfunction. Disruption of KIAA1440 led to embryonic lethality at the blastocyst stage. The high success rate of our approach demonstrates the rationale for the genome-wide functional examination of large proteins in mice using reverse genetics.

Animals↗