			Home About us Contact

Insertion Mutants (insertion + mutant)

Distribution by Scientific Domains

Life Sciences	82%
Medical Sciences	11%

Selected Abstracts

Molecular and Physiological Characterisation of an Insertion Mutant in the ARR21 Putative Response Regulator Gene from Arabidopsis thaliana

PLANT BIOLOGY, Issue 3 2003
J. Horák
Abstract: In our search for insertion mutants of Arabidopsis response regulator (ARR) genes, we identified a candidate for an ARR21 dSpm transposon insertion line in the SLAT collection by searching the SINS sequence database. Molecular characterisation of this line revealed that the transposon is integrated as a single copy 1727 bases downstream of the ATG signal, within the third intron of the ARR21 gene. The transposon insertion segregated in a Mendelian fashion from heterozygous plants that were allowed to self-pollinate. RT-PCR-based expression analysis showed that ARR21 transcript predominantly accumulates in siliques. In contrast, the full-length ARR21 transcript was not detectable in the ARR21 transposon insertion line, indicating that it harbours an insertion of dSpm transposon in the ARR21 gene. The ARR21 insertion mutant (arr21-1) was subjected to several physiological tests for a possible insertion-linked phenotype. However, our results revealed that the insertion in the ARR21 gene did not cause any alterations in viability and fertility, flowering time, sensitivity to ethylene, cytokinin or red light. We discuss these results in the light of recent findings about the function of the other members of the response regulator gene family of Arabidopsis. [source]

Cross-species divergence of the major recognition pathways of ubiquitylated substrates for ubiquitin/26S proteasome-mediated proteolysis

FEBS JOURNAL, Issue 3 2010
Antony S. Fatimababy
The recognition of ubiquitylated substrates is an essential element of ubiquitin/26S proteasome-mediated proteolysis (UPP), which is mediated directly by the proteasome subunit RPN10 and/or RPN13, or indirectly by ubiquitin receptors containing ubiquitin-like and ubiquitin-associated domains. By pull-down and mutagenesis assays, we detected cross-species divergence of the major recognition pathways. RPN10 plays a major role in direct recognition in Arabidopsis and yeast based on the strong affinity for the long and K48-linked ubiquitin chains. In contrast, both the RPN10 and RPN13 homologs play major roles in humans. For indirect recognition, the RAD23 and DSK2 homologs (except for the human DSK2 homolog) are major receptors. The human RAD23 homolog is targeted to the 26S proteasome by the RPN10 and RPN13 homologs. In comparison, Arabidopsis uses UIM1 and UIM3 of RPN10 to bind DSK2 and RAD23, respectively. Yeast uses UIM in RPN10 and LRR in RPN1. Overall, multiple proteasome subunits are responsible for the direct and/or indirect recognition of ubiquitylated substrates in yeast and humans. In contrast, a single proteasome subunit, RPN10, is critical for both the direct and indirect recognition pathways in Arabidopsis. In agreement with these results, the accumulation of ubiquitylated substrates and severe pleiotropic phenotypes of vegetative and reproductive growth are associated with the loss of RPN10 function in an Arabidopsis T-DNA insertion mutant. This implies that the targeting and proteolysis of the critical regulators involved are affected. These results support a cross-species mechanistic and functional divergence of the major recognition pathways for ubiquitylated substrates of UPP. Structured digital abstract ,,A list of the large number of protein-protein interactions described in this article is available via the MINT article ID MINT-7307429 [source]

The asgE locus is required for cell,cell signalling during Myxococcus xanthus development

MOLECULAR MICROBIOLOGY, Issue 4 2000
Anthony G. Garza
In response to starvation, Myxococcus xanthus undergoes a multicellular developmental process that produces a dome-shaped fruiting body structure filled with differentiated cells called myxospores. Two insertion mutants that block the final stages of fruiting body morphogenesis and reduce sporulation efficiency were isolated and characterized. DNA sequence analysis revealed that the chromosomal insertions are located in open reading frames ORF2 and asgE, which are separated by 68 bp. The sporulation defect of cells carrying the asgE insertion can be rescued phenotypically when co-developed with wild-type cells, whereas the sporulation efficiency of cells carrying the ORF2 insertion was not improved when mixed with wild-type cells. Thus, the asgE insertion mutant appears to belong to a class of developmental mutants that are unable to produce cell,cell signals required for M. xanthus development, but they retain the ability to respond to them when they are provided by wild-type cells. Several lines of evidence indicate that asgE cells fail to produce normal levels of A-factor, a cell density signal. A-factor consists of a mixture of heat-stable amino acids and peptides, and at least two heat-labile extracellular proteases. The asgE mutant yielded about 10-fold less heat-labile A-factor and about twofold less heat-stable A-factor than wild-type cells, suggesting that the primary defect of asgE cells is in the production or release of heat-labile A-factor. [source]

Possible role of the adhesin ace and collagen adherence in conveying resistance to disinfectants on Enterococcus faecalis

MOLECULAR ORAL MICROBIOLOGY, Issue 6 2008
G. Kayaoglu
Introduction:, This study aimed to evaluate whether the presence of the ace gene and Ace-mediated binding to collagen confers on Enterococcus faecalis resistance against common endodontic disinfectants. Methods:, Isogenic strains of E. faecalis: OG1RF (wild-type) and TX5256 (ace insertion mutant of OG1RF) were grown in brain,heart infusion broth at 46°C overnight. Standardized bacterial suspensions were pretreated for 1 h either with acid-soluble collagen or acidified phosphate-buffered saline (ac-PBS). Bacteria were challenged with chlorhexidine digluconate (CHX), iodine potassium-iodide (IKI), sodium hypochlorite (NaOCl), and calcium hydroxide [Ca(OH)2]. Samples were removed at 1, 3, and 6 h, and cultured on Todd,Hewitt agar plates. Colonies were counted, the absolute values were log transformed, and the data were statistically analyzed using Fisher's least significant differences test and t -test. Results:, OG1RF was more resistant than TX5256 to IKI, NaOCl, and Ca(OH)2 (P < 0.05). Collagen-exposed OG1RF was more resistant than the ac-PBS-pretreated OG1RF against CHX at 3 h and against IKI at 1 h (P < 0.05); no significant difference was found against NaOCl. As expected, the ace mutant strain, TX5256, pretreated with collagen or ac-PBS did not differ significantly in viability when challenged with CHX, IKI, and NaOCl. An unexpected result was found for Ca(OH)2: collagen-pretreated OG1RF and TX5256 were both more susceptible than ac-PBS-pretreated OG1RF and TX5256, respectively (P < 0.05). Conclusion:, The presence of the ace gene confers resistance against IKI, NaOCl, and Ca(OH)2 on E. faecalis. Exposure to collagen makes the wild-type bacterium more resistant against CHX and IKI; however, exposure to collagen apparently decreases resistance to Ca(OH)2. [source]

Isolation and characterization of the RAD54 gene from Arabidopsis thaliana

THE PLANT JOURNAL, Issue 6 2006
Keishi Osakabe
Summary Homologous recombination (HR) is an essential process in maintaining genome integrity and variability. In eukaryotes, the Rad52 epistasis group proteins are involved in meiotic recombination and/or HR repair. One member of this group, Rad54, belongs to the SWI2/SNF2 family of DNA-stimulated ATPases. Recent studies indicate that Rad54 has important functions in HR, both as a chromatin remodelling factor and as a mediator of the Rad51 nucleoprotein filament. Despite the importance of Rad54 in HR, no study of Rad54 from plants has yet been performed. Here, we cloned the full-length AtRAD54 cDNA sequence; an open reading frame of 910 amino acids encodes a protein with a predicted molecular mass of 101.9 kDa. Western blotting analysis showed that the AtRad54 protein was indeed expressed as a protein of approximately 110 kDa in Arabidopsis. The predicted protein sequence of AtRAD54 contains seven helicase domains, which are conserved in all other Rad54s. Yeast two-hybrid analysis revealed an interaction between Arabidopsis Rad51 and Rad54. AtRAD54 transcripts were found in all tissues examined, with the highest levels of expression in flower buds. Expression of AtRAD54 was induced by , -irradiation. A T-DNA insertion mutant of AtRAD54 devoid of full-length AtRAD54 expression was viable and fertile; however, it showed increased sensitivity to , -irradiation and the cross-linking reagent cisplatin. In addition, the efficiency of somatic HR in the mutant plants was reduced relative to that in wild-type plants. Our findings point to an important role for Rad54 in HR repair in higher plants. [source]

Helicobacter pylori mutagenesis by mariner in vitro transposition

FEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 2 2001
Betty P Guo
Abstract We have developed a method for generating transposon insertion mutants using mariner in vitro mutagenesis. The gene of interest was PCR-amplified and cloned. A kanamycin-marked mariner transposon was randomly inserted into the purified plasmid in an in vitro transposition reaction. After repair and propagation in Escherichia coli, purified mutagenized plasmid was introduced into Helicobacter pylori by natural transformation. Transformants were selected by plating on kanamycin. Mutants were predominantly the result of double homologous recombination, and multiple mutants (with insertions in distinct positions) were often obtained. The site of insertion was determined by PCR or sequencing. We have made mutations in known or potential virulence genes, including ureA, hopZ, and vacA, using kanamycin- and kanamycin/lacZ -marked transposons. Colonies carrying a kanamycin/lacZ transposon appeared blue on medium containing the chromogenic agent X-gal, allowing discrimination of mutant and wild-type H. pylori in mixed competition experiments. [source]

C-ring requirement in flagellar type III secretion is bypassed by FlhDC upregulation

MOLECULAR MICROBIOLOGY, Issue 2 2010
Marc Erhardt
Summary The cytoplasmic C-ring of the flagellum consists of FliG, FliM and FliN and acts as an affinity cup to localize secretion substrates for protein translocation via the flagellar-specific type III secretion system. Random T-POP transposon mutagenesis was employed to screen for insertion mutants that allowed flagellar type III secretion in the absence of the C-ring using the flagellar type III secretion system-specific hook,,-lactamase reporter (Lee and Hughes, 2006). Any condition resulting in at least a twofold increase in flhDC expression was sufficient to overcome the requirement for the C-ring and the ATPase complex FliHIJ in flagellar type III secretion. Insertions in known and unknown flagellar regulatory loci were isolated as well as chromosomal duplications of the flhDC region. The twofold increased flhDC mRNA level coincided in a twofold increase in the number of hook-basal bodies per cell as analysed by fluorescent microscopy. These results indicate that the C-ring functions as a nonessential affinity cup-like structure during flagellar type III secretion to enhance the specificity and efficiency of the secretion process. [source]

Genetic analysis of functions involved in the late stages of biofilm development in Burkholderia cepacia H111

MOLECULAR MICROBIOLOGY, Issue 2 2002
Birgit Huber
Summary Burkholderia cepacia and Pseudomonas aeruginosa often co-exist as mixed biofilms in the lungs of patients suffering from cystic fibrosis (CF). Here, we report the isolation of 13 random mini-Tn 5 insertion mutants of B. cepacia H111 that are defective in biofilm formation on a polystyrene surface. We show that the screening procedure used in this study is biased towards mutants defective in the late stages of biofilm development. A detailed quantitative analysis of the biofilm structures formed by wild-type and mutant strains revealed that the isolated mutants are impaired in their abilities to develop a typical three-dimensional biofilm structure. Molecular investigations showed that the genes required for biofilm maturation fall into several classes: (i) genes encoding for surface proteins; (ii) genes involved in the biogenesis and maintenance of an integral outer membrane; and (iii) genes encoding regulatory factors. It is shown that three of the regulatory mutants produce greatly reduced amounts of N -octanoylhomoserine lactone (C8-HSL). This compound serves as the major signal molecule of the cep quorum-sensing system. As this density-dependent regulatory system is involved in the regulation of biofilm maturation, we investigated the interplay between the three regulatory genes and the quorum-sensing cascade. The results of these investigations show that the identified genes encode for regulatory elements that are positioned upstream of the cep system, indicating that the quorum-sensing system of B. cepacia is a major checkpoint for biofilm formation. [source]

Molecular analysis of tyrosine- and phenylalanine-mediated repression of the tyrB promoter by the TyrR protein of Escherichia coli

MOLECULAR MICROBIOLOGY, Issue 5 2002
Ji Yang
Summary The mechanism of repression of the tyrB promoter by TyrR protein has been studied in vivo and in vitro. In tyrR+ strains, transcription of tyrB is repressed by either tyrosine or phenylalanine. Both of the TyrR binding sites (strong and weak TyrR boxes) lie downstream of the tyrB transcription start site and are required for tyrosine- or phenylalanine-mediated repression. Our results establish that the binding of the TyrR protein to the weak box, induced by cofactor tyrosine or phenylalanine, is critical for repression to occur. Neither the binding of the TyrR protein dimer formed in the presence of phenylalanine, nor the binding of the hexamer formed in the presence of tyrosine, blocks the binding of RNA polymerase to the promoter. Instead, open complex formation is inhibited in the presence of tyrosine whereas a step(s) following open complex formation is inhibited in the presence of phenylalanine. Moving the TyrR boxes 3 bp or more further away from the promoter affects tyrosine-mediated repression without affecting phenylalanine-mediated repression which remains unaltered until 6 bp are inserted between the TyrR boxes and the promoter. Analysis of deletion and insertion mutants fails to reveal any face of the helix specificity for either tyrosine- or phenylalanine-mediated repression. [source]

The asgE locus is required for cell,cell signalling during Myxococcus xanthus development

OsEF3, a homologous gene of Arabidopsis ELF3, has pleiotropic effects in rice

PLANT BIOLOGY, Issue 5 2009
C. Fu
Abstract Heading date is an important agronomic trait in rice. A rice mutant with a late heading date and no photoperiodic sensitivity in long or short day conditions was obtained from rice T-DNA insertion mutants in Zhonghua11 (ZH11). Through isolation and analysis of the flanking sequence of the T-NDA insertion site, the target sequence of insertion was obtained and found to locate in AP003296, the sequence accession number of rice chromosome 1 of RGP (http://rgp.dna.affrc.go.jp). The putative amino acid sequences of this target gene are homologous to the Arabidopsis protein ELF3 encoded by an early flowering gene. The rice target gene orthologous to Arabidopsis ELF3 is named OsEF3; this encodes a putative nematode responsive protein-like protein. OsEF3 has pleiotropic effects in rice that differ from the effects of Arabidopsis ELF3, which only affects biological rhythms. OsEF3 regulates heading date by influencing the BVG stage and does not affect photoperiodic sensitivity, which suggests that the OsEF3 gene may be involved in an autonomous pathway in rice. OsEF3 may affect root development and kilo-grain weight by delaying cell division or cell elongation. [source]

Molecular and Physiological Characterisation of an Insertion Mutant in the ARR21 Putative Response Regulator Gene from Arabidopsis thaliana

An , -amylase (At4g25000) in Arabidopsis leaves is secreted and induced by biotic and abiotic stress

PLANT CELL & ENVIRONMENT, Issue 4 2007
ELIZABETH A. DOYLE
ABSTRACT Leaves are reported to contain a secreted , -amylase that accumulates during senescence or after biotic or abiotic stress; however, a gene encoding this enzyme has not been described. Because a secreted amylase is isolated from plastidic starch, the function of this enzyme is difficult to predict, but circumstantial evidence suggests that it may degrade starch after cell death. The Arabidopsis thaliana genome contains three , -amylase genes, one of which, AMY1 (At4g25000), has a putative signal sequence suggesting that the protein may be secreted. Two independent T-DNA insertion mutants in AMY1 lacked an amylase band on starch zymograms, which was previously named ,A1'. Washed leaf protoplasts contained reduced A1 activity suggesting that the enzyme is secreted. Native AMY1, fused to a weakly fluorescent form of GFP, was sensitive to proteinase K infiltrated into leaf apoplastic spaces, while a cytosolic form of GFP was unaffected until cell breakage, confirming that the AMY1 protein is secreted. Amylase A1 was transcriptionally induced in senescing leaves and in leaves exposed to heat stress, treated with abscisic acid or infected with Pseudomonas syringae pv. tomato expressing avrRpm1. The A1 amylase was also extremely heat resistant and its expression was up-regulated in cpr5-2, an activated defence response mutant. [source]

Duplicated P5CS genes of Arabidopsis play distinct roles in stress regulation and developmental control of proline biosynthesis

THE PLANT JOURNAL, Issue 1 2008
Gyöngyi Székely
Summary ,-1-pyrroline-5-carboxylate synthetase enzymes, which catalyse the rate-limiting step of proline biosynthesis, are encoded by two closely related P5CS genes in Arabidopsis. Transcription of the P5CS genes is differentially regulated by drought, salinity and abscisic acid, suggesting that these genes play specific roles in the control of proline biosynthesis. Here we describe the genetic characterization of p5cs insertion mutants, which indicates that P5CS1 is required for proline accumulation under osmotic stress. Knockout mutations of P5CS1 result in the reduction of stress-induced proline synthesis, hypersensitivity to salt stress, and accumulation of reactive oxygen species. By contrast, p5cs2 mutations cause embryo abortion during late stages of seed development. The desiccation sensitivity of p5cs2 embryos does not reflect differential control of transcription, as both P5CS mRNAs are detectable throughout embryonic development. Cellular localization studies with P5CS,GFP gene fusions indicate that P5CS1 is sequestered into subcellular bodies in embryonic cells, where P5CS2 is dominantly cytoplasmic. Although proline feeding rescues the viability of mutant embryos, p5cs2 seedlings undergo aberrant development and fail to produce fertile plants even when grown on proline. In seedlings, specific expression of P5CS2,GFP is seen in leaf primordia where P5CS1,GFP levels are very low, and P5CS2,GFP also shows a distinct cell-type-specific and subcellular localization pattern compared to P5CS1,GFP in root tips, leaves and flower organs. These data demonstrate that the Arabidopsis P5CS enzymes perform non-redundant functions, and that P5CS1 is insufficient for compensation of developmental defects caused by inactivation of P5CS2. [source]

The Arabidopsis ClpB/Hsp100 family of proteins: chaperones for stress and chloroplast development

THE PLANT JOURNAL, Issue 1 2007
Ung Lee
Summary The Casein lytic proteinase/heat shock protein 100 (Clp/Hsp100) proteins are chaperones that act to remodel/disassemble protein complexes and/or aggregates using the energy of ATP. In plants, one of the best-studied proteins from this family is cytosolic ClpB1 (At1g74310), better known in Arabidopsis as AtHsp101, which is a heat shock protein required for acclimation to high temperatures. Three other ClpB homologues have been identified in the Arabidopsis genome (ClpB2, ClpB3 and ClpB4; At4g14670, At5g15450 and At2g25140). To define further the roles of these chaperones in plants we investigated their intracellular localization, evolutionary relationships, patterns of expression and the phenotypes of corresponding T-DNA insertion mutants. We first found that ClpB2 was misannotated; there is no functional ClpB/Hsp100 gene at this locus. By fusing the putative transit peptides of ClpB3 and ClpB4 with GFP, we showed that these proteins are targeted to the chloroplast and mitochondrion, respectively, and we therefore designated them as ClpB-p and ClpB-m. Phylogenetic analysis supports two major lineages of ClpB proteins in plants, an ,eukaryotic', cytosol/nuclear-localized group containing AtHsp101, and an organelle-localized lineage, containing both ClpB-p and ClpB-m. Although AtHsp101, ClpB-p and ClpB-m transcripts all accumulate dramatically at high temperatures, the T-DNA insertion mutants of ClpB-p and ClpB-m show no evidence of seedling heat stress phenotypes similar to those observed in AtHsp101 mutants. Strikingly, ClpB-p knockouts were seedling lethals, failing to accumulate chlorophyll or properly develop chloroplasts. Thus, in plants, the function of ClpB/Hsp100 proteins is not restricted to heat stress, but a specific member of the family provides housekeeping functions that are essential to chloroplast development. [source]

Rapid identification of Arabidopsis insertion mutants by non-radioactive detection of T-DNA tagged genes

THE PLANT JOURNAL, Issue 2 2002
Gabino Ríos
Summary To assist in the analysis of plant gene functions we have generated a new Arabidopsis insertion mutant collection of 90 000 lines that carry the T-DNA of Agrobacterium gene fusion vector pPCV6NFHyg. Segregation analysis indicates that the average frequency of insertion sites is 1.29 per line, predicting about 116 100 independent tagged loci in the collection. The average T-DNA copy number estimated by Southern DNA hybridization is 2.4, as over 50% of the insertion loci contain tandem T-DNA copies. The collection is pooled in two arrays providing 40 PCR templates, each containing DNA from either 4000 or 5000 individual plants. A rapid and sensitive PCR technique using high-quality template DNA accelerates the identification of T-DNA tagged genes without DNA hybridization. The PCR screening is performed by agarose gel electrophoresis followed by isolation and direct sequencing of DNA fragments of amplified T-DNA insert junctions. To estimate the mutation recovery rate, 39 700 lines have been screened for T-DNA tags in 154 genes yielding 87 confirmed mutations in 73 target genes. Screening the whole collection with both T-DNA border primers requires 170 PCR reactions that are expected to detect a mutation in a gene with at least twofold redundancy and an estimated probability of 77%. Using this technique, an M2 family segregating a characterized gene mutation can be identified within 4 weeks. [source]

Candida albicans protein kinase CK2 governs virulence during oropharyngeal candidiasis

CELLULAR MICROBIOLOGY, Issue 1 2007
Lisa Y. Chiang
Summary To identify Candida albicans genes whose proteins are necessary for host cell interactions and virulence, a collection of C. albicans insertion mutants was screened for strains with reduced capacity to damage endothelial cells in vitro. This screen identified CKA2. CKA2 and its homologue CKA1 encode the catalytic subunits of the protein kinase CK2. cka2,/cka2, strains of C. albicans were constructed and found to have significantly reduced capacity to damage both endothelial cells and an oral epithelial cell line in vitro. Although these strains invaded endothelial cells similarly to the wild-type strain, they were defective in oral epithelial cell invasion. They were also hypersusceptible to hydrogen peroxide, but not to high salt or to cell wall damaging agents. A cka1,/cka1, mutant caused normal damage to both endothelial cells and oral epithelial cells, and it was not hypersusceptible to hydrogen peroxide. However, overexpression of CKA1 in a cka2,/cka2, strain restored wild-type phenotype. Although the cka2,/cka2, mutant had normal virulence in the mouse model of haematogenously disseminated candidiasis, it had significantly attenuated virulence in the mouse model of oropharyngeal candidiasis. Therefore, Cka2p governs the interactions of C. albicans with endothelial and oral epithelial cells in vitro and virulence during oropharyngeal candidiasis. [source]