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At least 1,153 records · Page 64Linked to original sources
PubMed2003

Dissecting plant meiosis using Arabidopsis thaliana mutants.

Meiosis is a key stage in the life cycle of all sexually reproducing eukaryotes. In plants, specialized reproductive cells differentiate from somatic tissue. These cells then undergo a single round of DNA replication followed by two rounds of chromosome division to produce haploid cells that then undergo further rounds of mitotic division to produce the pollen grain and embryo sac. A detailed cytological description of meiosis has been built up over many years, based on studies in a wide range of plants. Until recently, comparable molecular studies have proved too challenging, however, a number of groups are beginning to use Arabidopsis thaliana to overcome this problem. A range of meiotic mutants affecting key stages in meiosis have been identified using a combination of screening for plants exhibiting reduced fertility and, more recently, using a reverse genetics approach. These are now providing the means to identify and characterize the activity of key meiotic genes in flowering plants.

Arabidopsis↗
PubMed2004

Using mutants to probe the in vivo function of plastid envelope membrane metabolite transporters.

During the last 15 years, much progress has been made in discovering genes encoding solute transporters of the inner plastid envelope membrane. For example, genes encoding transporters for phosphorylated intermediates, dicarboxylates, adenine nucleotides, inorganic anions, and monosaccharides have been cloned. In many cases, the corresponding proteins have been expressed in recombinant host systems for further functional studies, thus allowing detailed in vitro characterization of transporter properties. Knowledge of the gene sequences encoding these transporters have allowed reverse-genetic approaches to study transporter function in vivo. Antisense repression and T-DNA insertion mutagenesis have provided a range of transgenic and mutant plants in which the activity of specific plastid envelope transporters are massively decreased or abolished. Plants with altered transporter activities represent excellent tools to probe the in vivo function of these transporters. Moreover, changing the permeability of the plastid envelope membrane permits the targeted manipulation of subcellular metabolite pools.

Membrane Transport Proteins↗
PubMed2004

RNA interference in Agrobacterium rhizogenes-transformed roots of Arabidopsis and Medicago truncatula.

RNA interference (RNAi) is a powerful reverse genetic tool to study gene function. The data presented here show that Agrobacterium rhizogenes-mediated RNAi is a fast and effective tool to study genes involved in root biology. The Arabidopsis gene KOJAK, involved in root hair development, was efficiently knocked down. A. rhizogenes-mediated root transformation is a fast method to generate adventitious, genetically transformed roots. In order to select for co-transformed roots a binary vector was developed that enables selection based on DsRED1 expression, with the additional benefit that chimaeric roots can be discriminated. The identification of chimaeric roots provided the opportunity to examine the extent of systemic spread of the silencing signal in the composite plants of both Arabidopsis and Medicago truncatula. It is shown that RNA silencing does not spread systemically to non-co-transformed (lateral) roots and only inefficiently to the non-transgenic shoot. Furthermore, evidence is presented which shows that RNAi is cell autonomous in the root epidermis.

Arabidopsis↗
PubMed2004

Tomato fruit cuticular waxes and their effects on transpiration barrier properties: functional characterization of a mutant deficient in a very-long-chain fatty acid beta-ketoacyl-CoA synthase.

Cuticular waxes play a pivotal role in limiting transpirational water loss across the plant surface. The correlation between the chemical composition of the cuticular waxes and their function as a transpiration barrier is still unclear. In the present study, intact tomato fruits (Lycopersicon esculentum) are used, due to their astomatous surface, as a novel integrative approach to investigate this composition- function relationship: wax amounts and compositions of tomato were manipulated before measuring unbiased cuticular transpiration. First, successive mechanical and extractive wax-removal steps allowed the selective modification of epi- and intracuticular wax layers. The epicuticular film consisted exclusively of very-long-chain aliphatics, while the intracuticular compartment contained large quantities of pentacyclic triterpenoids as well. Second, applying reverse genetic techniques, a loss-of-function mutation with a transposon insertion in a very-long-chain fatty acid elongase beta-ketoacyl-CoA synthase was isolated and characterized. Mutant leaf and fruit waxes were deficient in n-alkanes and aldehydes with chain lengths beyond C30, while shorter chains and branched hydrocarbons were not affected. The mutant fruit wax also showed a significant increase in intracuticular triterpenoids. Removal of the epicuticular wax layer, accounting for one-third of the total wax coverage on wild-type fruits, had only moderate effects on transpiration. By contrast, reduction of the intracuticular aliphatics in the mutant to approximately 50% caused a 4-fold increase in permeability. Hence, the main portion of the transpiration barrier is located in the intracuticular wax layer, largely determined by the aliphatic constituents, but modified by the presence of triterpenoids, whereas epicuticular aliphatics play a minor role.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗
PubMed2005

Analysis of mutations induced by carbon ions in Arabidopsis thaliana.

To investigate the nature of mutations induced by accelerated ions in higher plants, the effects of carbon-ion-irradiation were compared with those of electron-irradiation in Arabidopsis thaliana. Point-like mutations and rearrangements were induced at a similar frequency after carbon-ion-irradiation, whereas point-like mutations were more frequently induced after electron-irradiation. Sequence analysis revealed that carbon-ion-induced point-like mutations were mostly short deletions. In the case of rearrangements, deletions, inversions, insertions, and translocations were found. The estimated frequency of deletion induction was comparable to that of fast neutrons. Analysis of chromosome breakpoints revealed that carbon ions frequently deleted small regions around the breakpoints, whereas electron-irradiation often duplicated these regions. Moreover, for both types of radiation, broken ends with microhomologies were frequently rejoined. Results of the breakpoint and broken end analyses suggest that non-homologous end-joining (NHEJ) leads to the rejoining of double strand breaks (dsbs) after cells are exposed to both types of radiation, but the type of NHEJ that occurs as a result of damage is different. The results indicated that carbon-ion-induced mutations are most likely nulls and that the induced rearrangements may arise through a unique mechanism. These findings indicate that accelerated ions are a useful mutagen for both forward and reverse genetics for plants.

Arabidopsis↗
PubMed2006

BIGYIN, an orthologue of human and yeast FIS1 genes functions in the control of mitochondrial size and number in Arabidopsis thaliana.

Reverse-genetics was used to evaluate the role of an Arabidopsis homologue of the human and yeast FIS1 genes, which are both involved in mitochondrial fission. Two independent T-DNA insertion mutants of gene At3g57090 were identified and genetically transformed to express mitochondria-targeted GFP to enable visualization of mitochondria in vivo. Plants homozygous for either of the recessive T-DNA mutant alleles, termed bigyin1-1 (bgy1-1) and bigyin1-2 (bgy1-2), displayed an abnormal mitochondrial morphology. Disruption of BIGYIN leads to a reduced number of mitochondria per cell, coupled to a large increase in the size of individual mitochondria, relative to wild-type. It is concluded that BIGYIN is an Arabidopsis FIS orthologue and is part of the Arabidopsis mitochondrial division apparatus.

Amino Acid Sequence↗
PubMed2006

Transcriptomics and functional genomics of plant defence induction by phloem-feeding insects.

The relationship between phloem-feeding insects (PFIs) and plants offers an intriguing example of a highly specialized biotic interaction. These insects have evolved to survive on a nutritionally imbalanced diet of phloem sap, and to minimize wound responses in their host plants. As a consequence, plant perception of and responses to PFIs differ from plant interactions with other insect-feeding guilds. Transcriptome-wide analyses of gene expression are currently being applied to characterize plant responses to PFIs in crop plants with race-specific innate resistance, as well as in compatible interactions with susceptible hosts. Recent studies indicate that PFIs induce transcriptional reprogramming in their host plants, and that plant responses to PFIs appear to be quantitatively and qualitatively different from responses to other insects or pathogens. Transcript profiling studies also suggest that PFIs induce cell wall modifications, reduce photosynthetic activity, manipulate source-sink relations, and modify secondary metabolism in their hosts, and many of these responses appear to occur within the phloem tissue. Plant responses to these insects appear to be regulated in part by the salicylate, jasmonate, and ethylene signalling pathways. As additional transcript profiling data become available, forward and reverse genetic approaches will be necessary to determine which changes in gene expression influence resistance or susceptibility to PFIs.

Animals↗
PubMed2006

Proteome changes in Arabidopsis thaliana roots upon exposure to Cd2+.

Cadmium is a major environmental pollutant that enters human food via accumulation in crop plants. Responses of plants to cadmium exposure--which directly influence accumulation rates--are not well understood. In general, little is known about stress-elicited changes in plants at the proteome level. Alterations in the root proteome of hydroponically grown Arabidopsis thaliana plants treated with 10 microM Cd(2+) for 24 h are reported here. These conditions trigger the synthesis of phytochelatins (PCs), glutathione-derived metal-binding peptides, shown here as PC2 accumulation. Two-dimensional gel electrophoresis using different pH gradients in the first dimension detected on average approximately 1100 spots per gel type. Forty-one spots indicated significant changes in protein abundance upon Cd(2+) treatment. Seventeen proteins found in 25 spots were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Selected results were independently confirmed by western analysis and selective enrichment of a protein family (glutathione S-transferases) through affinity chromatography. Most of the identified proteins belong to four different classes: metabolic enzymes such as ATP sulphurylase, glycine hydroxymethyltransferase, and trehalose-6-phosphate phosphatase; glutathione S-transferases; latex allergen-like proteins; and unknown proteins. These results represent a basis for reverse genetics studies to better understand plant responses to toxic metal exposure and to the generation of internal sinks for reduced sulphur.

Arabidopsis↗
PubMed2006

Loss of ovule identity induced by overexpression of the fertilization-related kinase 2 (ScFRK2), a MAPKKK from Solanum chacoense.

In order to gain information about protein kinases acting during plant fertilization and embryogenesis, a reverse genetic approach was used to determine the role of protein kinases expressed in reproductive tissues. Two cDNA clones named ScFRK1 and ScFRK2 (Solanum chacoense fertilization-related kinase 1 and 2) were isolated from an expressed sequence tag (EST) library normalized for weakly expressed genes in fertilized ovaries. These showed significant sequence similarities to members of the mitogen-activated protein kinase kinase kinase (MAPKKK) family. RNA gel blot and RNA in situ hybridization analyses confirmed the strong up-regulation of ScFRK2 in ovules after fertilization. In addition, ScFRK2 mRNAs accumulate during early ovule development in the megasporocyte and in the integument of developing ovules. Overexpression of ScFRK2 led to the production of fruits with a severely reduced number of seeds. The seeds that were produced also exhibited developmental retardation. Analysis of ovaries prior to fertilization showed that the seedless phenotype was caused by a homeotic conversion of ovules into carpel-like structures. The present observations are consistent with the role of ScFRK2 in pre- and post-fertilization events. Furthermore, overexpression of ScFRK2 led to changes in the expression of the class D floral homeotic gene ScFBP11, suggesting that the ScFRK2 kinase may interact, directly or indirectly, with the FBP7/11 pathway that directs establishment of ovule identity.

Amino Acid Sequence↗
PubMed1987

Precise and imprecise somatic excision of the transposon Tc1 in the nematode C. elegans.

Eleven chromosomal products of somatic excision of Tc1 transposable elements have been cloned and sequenced. The cloning method did not involve genetic reversion; therefore the products analyzed should be representative. Six empty religated target sites were from excision of one Tc1 element inserted near actin genes on linkage group V; five were from a second Tc1 element inserted elsewhere on the same linkage group. All six products from the first element were identical in sequence to an empty target site from a second strain, indicating excision had been precise. Two of the products from the second element were also precise, whereas the other three contained four extra nucleotides at the point of excision, indicating an imprecise excision. The four nucleotides are the same in all cases and could represent two terminal nucleotides of the transposon plus a two-nucleotide target site duplication. The difference in the ratio of precise to imprecise excision at the two insertion sites suggests a possible chromosomal position effect on the pathway of Tc1 somatic excision.

Animals↗
PubMed1988

Conservation of short patches of amino acid sequence amongst proteins with a common function but evolutionarily distinct origins: implications for cloning genes and for structure-function analysis.

Small patches of identical amino acid sequences commonly occur in proteins that have the same function but are derived from evolutionarily distant organisms. Reverse translation of such patches into degenerate pools of oligonucleotides provide useful hybridization probes for cloning the gene for the corresponding protein from other organisms. Since the conserved patches of identical amino acid sequence are probably important for the protein's biological function, they are preferred targets for reverse genetic studies aimed at defining structure-function relationships.

Amino Acid Sequence↗
PubMed1991

Transgenic expression of aminoglycoside adenine transferase in the chloroplast: a selectable marker of site-directed transformation of chlamydomonas.

Expression vectors for Chlamydomonas reinhardtii chloroplast transformation have been constructed with transcription and translation signals from chloroplast genes. The bacterial aadA sequence, coding for aminoglycoside 3" adenyl transferase, was inserted in these vectors and introduced into the C. reinhardtii chloroplast by particle gun transformation. The stable transgenic expression of this foreign protein in the chloroplast confers spectinomycin and streptomycin resistance to the transformed cells. This new marker can be used as a reporter of gene expression, and as a portable selectable cassette for chloroplast reverse genetics. Targetted gene disruption mutants of loci required for photosynthesis, tscA and psaC, were thus obtained. A gene disruption of an unidentified open reading frame, ORF472, remained heteroplasmic, suggesting that it has a vital function.

Base Sequence↗
PubMed1994

Gene expression mediated by bacteriophage T3 and T7 RNA polymerases in transgenic trypanosomes.

Messenger RNAs of higher eukaryotes share a functionally essential 5' monomethyl CAP structure generated during a reaction that is linked exclusively to RNA polymerase II transcription. In unicellular parasites belonging to the Kinetoplastida, however, mRNAs acquire their 5' CAP through a trans-splicing reaction which effectively uncouples pol II transcription and capping. Consequently functional mRNAs can be produced by endogenous RNA polymerase I. Here we demonstrate the extension of this flexibility to heterologous bacteriophage polymerases. Transgenic Trypanosoma brucei cell lines stably expressing functional, nuclearly localized T3 or T7 RNA polymerase were established and assayed using reporter plasmids bearing the corresponding phage promoters. In these cell lines the levels of phage promoter-driven gene expression ranges from one half to greater than 5 times that mediated by endogenous pol I. Analysis of 5' ends of transcripts synthesized by the T7 polymerase revealed that they are trans-spliced. Thus the usual eukaryotic link between mRNA production and pol II transcription can be by-passed by the introduced phage polymerases, thereby significantly expanding the critically small panel of promoters currently available for exploitation in reverse genetic approaches in T. brucei.

Animals↗
PubMed2002

RNA interference in mammalian cells using siRNAs synthesized with T7 RNA polymerase.

Methods that allow the specific silencing of a desired gene are invaluable tools for research. One of these is based on RNA interference (RNAi), a process by which double-stranded RNA (dsRNA) specifically suppresses the expression of a target mRNA. Recently, it has been reported that RNAi also works in mammalian cells if small interfering RNAs (siRNAs) are used to avoid activation of the interferon system by long dsRNA. Thus, RNAi could become a major tool for reverse genetics in mammalian systems. However, the high cost and the limited availability of the short synthetic RNAs and the lack of certainty that a designed siRNA will work present major drawbacks of the siRNA technology. Here we present an alternative method to obtain cheap and large amounts of siRNAs using T7 RNA polymerase. With multiple transfection procedures, including calcium phosphate co-precipitation, we demonstrate silencing of both exogenous and endogenous genes.

Cell Line↗
PubMed1981

Structure and expression of a cloned beta o thalassaemic globin gene.

We have cloned the single beta-globin gene from an Italian patient who is a double heterozygote for beta o/delta beta o thalassaemia. RNA isolated from nucleated red cells from this patient can be translated in vitro to give detectable levels of A gamma- G gamma and alpha-globin, but no beta-globin. S1-mapping transcription studies show that beta-globin mRNA is present at about 1-3% of the level of alpha- and gamma-globin mRNA. In addition, the expression of this gene has been studied by reversed genetics. SV40-plasmid-beta o-globin gene recombinants have been transfected into Hela cells and analysed for beta-globin mRNA. In contrast to the results obtained with mRNA isolated directly from the blood of this patient, in the transfected Hela cells the same level of beta-globin mRNA is seen for the beta o thalassaemic globin gene and for a normal beta-globin gene. To elucidate the nature of the lesion, the entire DNA sequence of the beta-globin gene of this patient has been determined. The sequence shows that this gene contains a termination codon at position 39 (CAG - greater than UAG). Otherwise, there is a remarkable conservation of the entire DNA sequence.

Base Sequence↗
PubMed2002

Structural variation and functional importance of a D-loop-T-loop interaction in valine-accepting tRNA-like structures of plant viral RNAs.

Valine-accepting tRNA-like structures (TLSs) are found at the 3' ends of the genomic RNAs of most plant viruses belonging to the genera Tymovirus, Furovirus, Pomovirus and Pecluvirus, and of one Tobamovirus species. Sequence alignment of these TLSs suggests the existence of a tertiary D-loop-T-loop interaction consisting of 2 bp, analogous to those in the elbow region of canonical tRNAs. The conserved G(18).Psi(55) pair of regular tRNAs is found to covary in these TLSs between G.U (possibly also modified to G.Psi) and A.G. We have mutated the relevant bases in turnip yellow mosaic virus (TYMV) and examined the mutants for symptom development on Chinese cabbage plants and for accumulation of genetic reversions. Development of symptoms is shown to rely on the presence of either A.G or G.U in the original mutants or in revertants. This finding supports the existence and functional importance of this tertiary interaction. The fact that only G.U and A.G are accepted at this position appears to result from steric and energetic limitations related to the highly compact nature of the elbow region. We discuss the implications of these findings for the various possible functions of the valine-accepting TLS.

Base Sequence↗
PubMed2002

The fission yeast pfh1(+) gene encodes an essential 5' to 3' DNA helicase required for the completion of S-phase.

The Cdc24 protein plays an essential role in chromosomal DNA replication in the fission yeast Schizosaccharomyces pombe, most likely via its direct interaction with Dna2, a conserved endonuclease-helicase protein required for Okazaki fragment processing. To gain insights into Cdc24 function, we isolated cold-sensitive chromosomal suppressors of the temperature-sensitive cdc24-M38 allele. One of the complementation groups of such suppressors defined a novel gene, pfh1(+), encoding an 805 amino acid nuclear protein highly homologous to the Saccharomyces cerevisiae Pif1p and Rrm3p DNA helicase family proteins. The purified Pfh1 protein displayed single-stranded DNA-dependent ATPase activity as well as 5' to 3' DNA helicase activity in vitro. Reverse genetic analysis in S.pombe showed that helicase activity was essential for the function of the Pfh1 protein in vivo. Schizosaccharomyces pombe cells carrying the cold-sensitive pfh1-R20 allele underwent cell cycle arrest in late S/G2-phase of the cell cycle when shifted to the restrictive temperature. This arrest was dependent upon the presence of a functional late S/G2 DNA damage checkpoint, suggesting that Pfh1 is required for the completion of DNA replication. Furthermore, at their permissive temperature pfh1-R20 cells were highly sensitive to the DNA-alkylating agent methyl methanesulphonate, implying a further role for Pfh1 in the repair of DNA damage.

Adenosine Triphosphatases↗
PubMed2003

A novel engineered meganuclease induces homologous recombination in yeast and mammalian cells.

Homologous gene targeting is the ultimate tool for reverse genetics, but its use is often limited by low efficiency. In a number of recent studies, site- specific DNA double-strand breaks (DSBs) have been used to induce efficient gene targeting. Engineering highly specific, dedicated DNA endonucleases is the key to a wider usage of this technology. In this study, we present two novel, chimeric meganucleases, derived from homing endonucleases. The first one is able to induce recombination in yeast and mammalian cells, whereas the second cleaves a novel (chosen) DNA target site. These results are a first step toward the generation of custom endonucleases for the purpose of targeted genome engineering.

Animals↗