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

A bacterial conjugation machinery recruited for pathogenesis.

Type IV secretion systems (T4SS) are multicomponent transporters of Gram-negative bacteria adapted to functions as diverse as DNA transfer in bacterial conjugation or the delivery of effector proteins into eukaryotic target cells in pathogenesis. The generally modest sequence conservation between T4SS may reflect their evolutionary distance and/or functional divergence. Here, we show that the establishment of intraerythrocytic parasitism by Bartonella tribocorum requires a putative T4SS, which shares an unprecedented level of sequence identity with the Trw conjugation machinery of the broad-host-range antibiotic resistance plasmid R388 (up to 80% amino acid identity for individual T4SS components). The highly conserved T4SS loci are collinear except for the presence of numerous tandem gene duplications in B. tribocorum, which mostly encode variant forms of presumed surface-exposed pilus subunits. Conservation is not only structural, but also functional: R388 mutated in either trwD or trwH encoding essential T4SS components could be trans-complemented for conjugation by the homologues of the B. tribocorum system. Conservation also includes the transcription regulatory circuit: both T4SS loci encode a highly homologous and interchangeable KorA/KorB repressor system that negatively regulates the expression of all T4SS components. This striking example of adaptive evolution reveals the capacity of T4SS to assume dedicated functions in either DNA transfer or pathogenesis over rather short evolutionary distance and implies a novel role for the conjugation systems of widespread broad-host-range plasmids in the evolution of bacterial pathogens.

Bartonella↗
PubMed1997

Cell-cycle-regulated transcription of A- and B-type plant cyclin genes in synchronous cultures.

Synchronously dividing cell cultures of Catharanthus roseus were used to isolate cDNAs for two mitotic cyclins, named CYS and CYM. The deduced protein sequence of CYS is similar to that of A-type cyclins, and CYM belongs to the group of B-type cyclins. In a fashion similar to the pattern of expression seen for A-type and B-type cyclins in mammalian cells, CYS is expressed before CYM in C. roseus cells during the cell cycle. CYS mRNA accumulated at the onset of S phase and disappeared early in the G2 phase, whereas CYM mRNA was detected in the G2 and M phases of the cell cycle. Tobacco homologs of the two genes showed similar cell-cycle dependent expression patterns in synchronous cultures of tobacco BY2 cells. In both systems, CYS was expressed much earlier in the cell cycle than most other plant A-type cyclins, and hence CYS along with the soybean cyc1GM can be classified into a distinct subclass. The activities of CYM and CYS promoters during the cell cycle were analyzed in stably transformed tobacco BY2 cells. Cyclin promoter sequences of 0.5 kb could confer the typical cell-cycle-dependent expression to the beta-glucuronidase (GUS) reporter gene: the CYS promoter directed S-phase-specific expression, whereas the CYM promoter drove M-phase-specific expression. These results indicate the important role of transcriptional regulation in the oscillations of cyclin mRNA levels during the cell cycle.

Cell Cycle↗
PubMed1997

adg2-1 represents a missense mutation in the ADPG pyrophosphorylase large subunit gene of Arabidopsis thaliana.

Arabidopsis mutants affecting ADPG pyrophosphorylase (ADGase) activity can be divided into two complementation groups, adg1 and adg2. Previous biochemical studies of adg2-1 mutant indicated that mutant plants do not accumulate ADGase large subunit protein and that ADGase small subunits assemble as homotetramers. This suggested that the ADG2 gene may encode the large subunit of ADGase. In this paper, it is shown that adg2-1 mutant plants accumulate near wild-type levels of transcripts encoding both the large and small subunits of ADGase. However, by RFLP analysis and complementation of adg2-1 with the ADGase large subunit gene, we show that the adg2-1 mutant does represent a mutation of the ADGase large subunit gene. Sequence analysis of the adg2-1 allele revealed a missense mutation. The results therefore suggest either that the missense mutation affects the stability of the ADGase large subunit protein or that it prevents assembly of the large subunit into holoenzyme.

Amino Acid Sequence↗
PubMed2002

Development of a GFP reporter gene for Chlamydomonas reinhardtii chloroplast.

Reporter genes have been successfully used in chloroplasts of higher plants, and high levels of recombinant protein expression have been reported. Reporter genes have also been used in the chloroplast of Chlamydomonas reinhardtii, but in most cases the amounts of protein produced appeared to be very low. We hypothesized that the inability to achieve high levels of recombinant protein expression in the C. reinhardtii chloroplast was due to the codon bias seen in the C. reinhardtii chloroplast genome. To test this hypothesis, we synthesized a gene encoding green fluorescent protein (GFP) de novo, optimizing its codon usage to reflect that of major C. reinhardtii chloroplast-encoded proteins. We monitored the accumulation of GFP in C. reinhardtii chloroplasts transformed with the codon-optimized GFP cassette (GFPct), under the control of the C. reinhardtii rbcL 5'- and 3'-UTRs. We compared this expression with the accumulation of GFP in C. reinhardtii transformed with a non-optimized GFP cassette (GFPncb), also under the control of the rbcL 5'- and 3'-UTRs. We demonstrate that C. reinhardtii chloroplasts transformed with the GFPct cassette accumulate approximately 80-fold more GFP than GFPncb-transformed strains. We further demonstrate that expression from the GFPct cassette, under control of the rbcL 5'- and 3'-UTRs, is sufficiently robust to report differences in protein synthesis based on subtle changes in environmental conditions, showing the utility of the GFPct gene as a reporter of C. reinhardtii chloroplast gene expression.

Amino Acid Sequence↗
PubMed1998

Molecular cloning and mutational analysis of the ddsA gene encoding decaprenyl diphosphate synthase from Gluconobacter suboxydans.

Decaprenyl diphosphate (decaprenyl-PP) synthase catalyzes the consecutive condensation of isopentenyl diphosphate with allylic diphosphates to produce decaprenyl-PP, which is used for the side chain of ubiquinone (Q)-10. We have cloned the synthase gene, designated ddsA, from Gluconobacter suboxydans and expressed it in Escherichia coli. Sequence analysis revealed the presence of an ORF of 948 bp capable of encoding a 33,898-Da polypeptide that displays high similarity (30-50%) to other prenyl diphosphate synthases. Expression of the ddsA gene complemented the lethality resulting from a defect in the octaprenyl diphosphate synthase gene of E. coli and produced Q-10, indicating that Q-10 can substitute for the function of Q-8. The His-tagged DdsA protein was purified to characterize its enzymatic properties. This enzyme required detergent (0.05% Triton X-100) and 10 mM Mg2+, for full activity. The Michaelis constants for geranyl diphosphate, all-E-farnesyl diphosphate and all-E-geranylgeranyl diphosphate were 7.00, 0.50 and 0.32 microM, respectively. Nine single-amino-acid substitutions were introduced upstream of conserved region II or VI. Most of the mutants showed a considerable decrease in catalytic activity or shortening of the ultimate chain length. However, the A70G mutant produced a longer-chain-length product than wild-type decaprenyl-PP synthase, and the A70Y mutant completely abolished the decaprenyl-PP synthase function, indicating that Ala70 is important for enzyme activity and the determination of the chain-length properties of DdsA.

Acetobacteraceae↗
PubMed1999

Adaptation of Pseudomonas fluorescens to the plant rhizosphere.

Saprophytic Pseudomonas are common root-colonizing bacteria that can improve plant health. Efficient exploitation of these bacteria in agriculture requires knowledge of traits that enhance ecological performance in the rhizosphere. Here, I describe the development and application of a promoter-trapping technology (IVET) that enables the isolation of Pseudomonas fluorescens genes that show elevated levels of expression in the rhizosphere. Using IVET, 20 P. fluorescens genes were identified that are induced during rhizosphere colonization, and their patterns of expression were analysed in laboratory media and in the rhizosphere. Fourteen genes showed significant homology to sequences in GenBank that are involved in nutrient acquisition, stress response, or secretion; six showed no homology. Seven of the rhizosphere-induced (rhi) genes have homology to known non-Pseudomonas genes. One of the rhi genes (hrcC) is a component of a type III secretion pathway, not previously known in non-parasitic bacteria. Together, these genes provide a view of the rhizosphere environment as perceived by a rhizosphere colonist, and suggest that the nature of the association between P. fluorescens and the plant root may be more complex and intimate than previously thought.

Adaptation, Physiological↗
PubMed1998

Optimization of genome search strategies for homozygosity mapping: influence of marker spacing on power and threshold criteria for identification of candidate regions.

Mapping of genes involved in rare recessive diseases is usually difficult because of the lack of families with more than one affected progeny. The problem may be avoided by using inbred affected individuals and the strategy of homozygosity mapping. In practice, the use of homozygosity mapping in a genome-wide scan requires that a set of markers regularly spaced and spanning the whole genome are tested. Investigators are then faced to the problem of choosing the spacing of markers. To help solve this problem, we give some useful clues by computing (1) the expected length of the region of identity by descent around the disease locus, (2) the distribution, given the spacing of markers, of the number of affected individuals expected not to be homozygous at the marker closest to the disease locus and, (3) the expected type-one error. We show that even if the markers are very closely spaced, it is not unlikely that some affected individuals in the sample will not be homozygous at the marker closest to the disease locus. Excluding a region by the criterion that all affected individuals in the sample are not homozygous may then dramatically increase the rate of false negatives. We thus propose to relax the criterion to declare a region candidate, based on the sample size and the spacing of markers.

Chromosome Mapping↗
PubMedDate not supplied

A RT-PCR assay for the rapid recognition of border disease virus.

A reverse transcription--polymerase chain reaction (RT-PCR) method was developed for the specific detection of border disease virus (BDV), using the primers PBD1 and PBD2 flanking a 225 bp DNA fragment, selected from the 5'noncoding region of the pestivirus genome. In tests on 70 pestiviruses it was shown to be BDV-specific. A closed, one-tube nested RT-PCR method employing general pestivirus outer primers (324 and 326), and the same BDV-specific inner primers (PBD1 and PBD2) in conjunction with a BDV-specific fluorogenic TaqMan probe also detected only BDV and was more sensitive. BDV-specific RT-PCR was used in combination with a PCR specific for bovine viral diarrhoea virus type 2 (BVDV2) to ascertain whether virus stocks contained mixtures of BDV and BVDV2. It was shown that the ovine pestivirus strains 175375 and 59386 were originally BDV, but after subculture had become contaminated with BVDV2. This explains a previously reported discrepancy in the genetic typing of 59386. Although the BDV-specific RT-PCR can also detect BDV in clinical samples, the assay is likely to be most useful for the rapid typing of laboratory pestivirus strains.

Animals↗
PubMedDate not supplied

Detection of bovine respiratory syncytial virus in experimentally infected balb/c mice.

The present study used an RT-nested-PCR and an immunohistochemistry assay to detect bovine respiratory syncytial virus in tissues from experimentally infected balb/c mice. As a first step, Chicken Embryo Related (CER) cell monolayers infected with the BRSV-25-BR strain isolated in Brazil were used for antigen production. Then, the infected lung and tracheal tissues of female balb/c mice were collected on 3, 5, 7 and 10 days post-infection and submitted to both techniques. Primers specific to F and G genes that amplify fragments of 481 bp and 371 bp, respectively, were used. The BRSV detection was not successful in all of the animals tested. The genomic fragment of the G gene from the organs of some infected mice on all analyzed post-infection days was amplified. However, in the RT-nested-PCR corresponding to the F gene, it was not possible to observe any amplified fragment. This was probably due to the higher sensitivity of the developed technique to amplify the fragment corresponding to the G gene compared to the F gene. Moreover, only three of the lungs collected five days post-infection were positive by immunohistochemistry. To the author's knowledge, this is the first study reporting bovine respiratory syncytial virus detection in balb/c mice after experimental inoculation.

Animals↗
PubMed2004

Self/nonself recognition in ascidian fertilization: vitelline coat protein HrVC70 is a candidate allorecognition molecule.

Ascidians are hermaphrodites releasing sperm and eggs simultaneously, but many species are self-sterile because of a self/nonself-recognition system in spermegg interaction. Here, we show that a 70-kDa vitelline coat protein, HrVC70, consisting of 12 epidermal growth factor-like repeats, plays a key role in self/nonself recognition during ascidian fertilization. We discovered that the amount of HrVC70 of the self-sterile mature oocytes is markedly higher than that of the self-fertile immature oocytes and that the selfsterile mature oocytes become self-fertile by acid treatment, which is able to release the HrVC70 from isolated vitelline coats. In addition, fertilization is strongly inhibited by the pretreatment of sperm with HrVC70 from a different individual, but not from the same individual, and the number of nonself sperm bound to HrVC70-agarose was significantly higher than that of self-sperm. A sequence analysis of HrVC70 disclosed that several amino acid residues in a restricted region are substituted at an individual level, with no identical sequences among the 10 individuals tested. Furthermore, genomic DNA analysis revealed that the epidermal growth factor-like domains correspond to the exons, and each intron is highly conserved among even- and odd-numbered introns, suggesting that multiple gene duplications or amplification of this region might have taken place during evolution. It was also found that diversity in cDNA sequences is derived from genomic DNA polymorphism probably elicited by crossing over and specific nucleotide substitutions. These results indicate that HrVC70 is a candidate allogeneic recognition molecule in the gamete interaction of the ascidian Halocynthia roretzi.

Amino Acid Sequence↗
PubMed2005

NF-kappaB activation and potentiation of proinflammatory responses by the Helicobacter pylori CagA protein.

The Helicobacter pylori immunodominant protein, CagA, is associated with severe gastritis and carcinoma. Injection of CagA into gastric epithelial cells by type IV secretion leads to actin-cytoskeletal rearrangements and cell scattering. CagA has been reported to have no role in the induction of transcription factor NF-kappaB and IL-8, which are crucial determinants for chronic inflammation. Here, we provide several lines of evidence showing that CagA is able to induce IL-8 in a time- and strain-dependent manner. We also show that by exchanging specific cagA genes, high IL-8-inducing H. pylori strains could be converted into low inducing strains and vice versa. Our results suggest that IL-8 release induced by CagA occurs via a Ras-->Raf-->Mek-->Erk-->NF-kappaB signaling pathway in a Shp-2- and c-Met-independent manner. Thus, CagA is a multifunctional protein capable of effecting both actin remodeling and potentiation of chemokine release.

Actins↗
PubMed2005

Why highly expressed proteins evolve slowly.

Much recent work has explored molecular and population-genetic constraints on the rate of protein sequence evolution. The best predictor of evolutionary rate is expression level, for reasons that have remained unexplained. Here, we hypothesize that selection to reduce the burden of protein misfolding will favor protein sequences with increased robustness to translational missense errors. Pressure for translational robustness increases with expression level and constrains sequence evolution. Using several sequenced yeast genomes, global expression and protein abundance data, and sets of paralogs traceable to an ancient whole-genome duplication in yeast, we rule out several confounding effects and show that expression level explains roughly half the variation in Saccharomyces cerevisiae protein evolutionary rates. We examine causes for expression's dominant role and find that genome-wide tests favor the translational robustness explanation over existing hypotheses that invoke constraints on function or translational efficiency. Our results suggest that proteins evolve at rates largely unrelated to their functions and can explain why highly expressed proteins evolve slowly across the tree of life.

Computational Biology↗
PubMed2002

Arabidopsis disrupted in SQD2 encoding sulfolipid synthase is impaired in phosphate-limited growth.

The sulfolipid sulfoquinovosyldiacylglycerol is one of the three nonphosphorous glycolipids that provide the bulk of the structural lipids in photosynthetic membranes of seed plants. Unlike the galactolipids, sulfolipid is anionic at physiological pH because of its 6-deoxy-6-sulfonate-glucose (sulfoquinovose) head group. The biosynthesis of this lipid proceeds in two steps: first, the assembly of UDP-sulfoquinovose from UDP-glucose and sulfite, and second, the transfer of the sulfoquinovose moiety from UDP-sulfoquinovose to diacylglycerol. The first reaction is catalyzed by the SQD1 protein in Arabidopsis. Here we describe the identification of the SQD2 gene of Arabidopsis. We propose that this gene encodes the sulfoquinovosyltransferase catalyzing the second step of sulfolipid biosynthesis. Expression of SQD1 and SQD2 in Escherichia coli reconstituted plant sulfolipid biosynthesis in this bacterium. Insertion of a transfer DNA into this gene in Arabidopsis led to complete lack of sulfolipid in the respective sqd2 mutant. This mutant showed reduced growth under phosphate-limited growth conditions. The results support the hypothesis that sulfolipid can function as a substitute of anionic phospholipids under phosphate-limited growth conditions. Along with phosphatidylglycerol, sulfolipid contributes to maintaining a negatively charged lipid-water interface, which presumably is required for proper function of photosynthetic membranes.

Arabidopsis↗
PubMed2001

Tomato Ve disease resistance genes encode cell surface-like receptors.

In tomato, Ve is implicated in race-specific resistance to infection by Verticillium species causing crop disease. Characterization of the Ve locus involved positional cloning and isolation of two closely linked inverted genes. Expression of individual Ve genes in susceptible potato plants conferred resistance to an aggressive race 1 isolate of Verticillium albo-atrum. The deduced primary structure of Ve1 and Ve2 included a hydrophobic N-terminal signal peptide, leucine-rich repeats containing 28 or 35 potential glycosylation sites, a hydrophobic membrane-spanning domain, and a C-terminal domain with the mammalian E/DXXXLphi or YXXphi endocytosis signals (phi is an amino acid with a hydrophobic side chain). A leucine zipper-like sequence occurs in the hydrophobic N-terminal signal peptide of Ve1 and a Pro-Glu-Ser-Thr (PEST)-like sequence resides in the C-terminal domain of Ve2. These structures suggest that the Ve genes encode a class of cell-surface glycoproteins with receptor-mediated endocytosis-like signals and leucine zipper or PEST sequences.

Amino Acid Sequence↗
PubMed2002

A cytosine methyltransferase homologue is essential for repeat-induced point mutation in Neurospora crassa.

During sexual development, Neurospora crassa inactivates genes in duplicated DNA segments by a hypermutation process, repeat-induced point mutation (RIP). RIP introduces C:G to T:A transition mutations and creates targets for subsequent DNA methylation in vegetative tissue. The mechanism of RIP and its relationship to DNA methylation are not fully understood. Mutations in DIM-2, a DNA methyltransferase (DMT) responsible for all known cytosine methylation in Neurospora, does not prevent RIP. We used RIP to disrupt a second putative DMT gene in the Neurospora genome and tested mutants for defects in DNA methylation and RIP. No effect on DNA methylation was detected in the tissues that could be assayed, but the mutants showed recessive defects in RIP. Duplications of the am and mtr genes were completely stable in crosses homozygous for the mutated potential DMT gene, which we call rid (RIP defective). The same duplications were inactivated normally in heterozygous crosses. Disruption of the rid gene did not noticeably affect fertility, growth, or development. In contrast, crosses homozygous for a mutation in a related gene in Ascobolus immersus, masc1, reportedly fail to develop and heterozygous crosses reduce methylation induced premeiotically [Malagnac, F., Wendel, B., Goyon, C., Faugeron, G., Zickler, D., et al. (1997) Cell 91, 281-290]. We isolated homologues of rid from Neurospora tetrasperma and Neurospora intermedia to identify conserved regions. Homologues possess all motifs characteristic of eukaryotic DMTs and have large distinctive C- and N-terminal domains.

Amino Acid Sequence↗