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P Dion

Publications and source records attributed to P Dion.

At least 19 recordsLinked to original sources
PubMed2001

Oligomerization of polyalanine expanded PABPN1 facilitates nuclear protein aggregation that is associated with cell death.

Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disorder characterized by progressive eyelid drooping, swallowing difficulties and proximal limb weakness. The autosomal dominant form of this disease is caused by short expansions of a (GCG)(6) repeat to (GCG)(8-13) in the PABPN1 gene, which results in the expansion of a polyalanine stretch from 10 to 12-17 alanines in the N-terminus of the protein. Mutated PABPN1 (mPABPN1) is able to induce nuclear protein aggregation and form filamentous nuclear inclusions, which are the pathological hallmarks of OPMD. PABPN1, when bound to poly(A) RNA, forms both linear filaments and discrete-sized, compact oligomeric particles in vitro. In the absence of poly(A) RNA, PABPN1 can form oligomers. Here we report that: (i) oligomerization of PABPN1 is mediated by two potential oligomerization domains (ODs); (ii) inactivating oligomerization of mPABPN1 by deletions of 6-8 amino acids in either of the ODs prevents nuclear protein aggregation; (iii) expression of mPABPN1 in COS-7 cells is associated with cell death; and (iv) preventing nuclear protein aggregation by inactivating oligomerization of mPABPN1 significantly reduces cell death. These findings suggest that oligomerization of PABPN1 plays a crucial role in the formation of OPMD nuclear protein aggregation, while the expanded polyalanine stretch is necessary but not sufficient to induce OPMD protein aggregation, and that the nuclear protein aggregation might be toxic and cause cell death. These observations also imply that inactivation of oligomerization of mPABPN1 might be a useful therapeutic strategy for OPMD.

Animals↗
PubMed2001

Schwannomin isoform-1 interacts with syntenin via PDZ domains.

The neurofibromatosis type 2 gene (NF2) is involved in the pathogenesis of benign tumors of the human nervous system. The NF2 protein, called schwannomin or merlin, is inactivated in virtually all schwannomas and meningiomas. The molecular mechanisms by which schwannomin functions as a tumor suppressor is unknown but believed to involve plasma membrane-cytoskeletal interactions. Two major alternatively spliced isoforms of schwannomin differing in their C termini have been reported. Using the yeast two-hybrid system, we have identified syntenin as a binding partner for schwannomin isoform-1 (sch-1). Syntenin is an adapter protein that couples transmembrane proteoglycans to cytoskeletal components and is involved in intracellular vesicle transport. The C terminus 25 amino acids of sch-1 and the two PDZ domains of syntenin mediate their binding, and mutations introduced within the VAFFEEL region of sch-1 defined a sequence crucial for syntenin recognition. We have showed that the two proteins interacted in vitro and in vivo and localized underneath the plasma membrane. Fibroblast cells expressing heterologous antisense syntenin display alterations in the subcellular distribution of sch-1. Together, these results provide the first functional clue to the existence of schwannomin isoforms and could unravel novel pathways for the transport and subcellular localization of schwannomin in vivo.

3T3 Cells↗
PubMed2000

CAG tract of MJD-1 may be prone to frameshifts causing polyalanine accumulation.

Machado-Joseph disease (MJD) is one of several disorders caused by the expansion of a coding CAG repeat (exp-CAG). The presence of intranuclear inclusions (INIs) in patients and cellular models of exp-CAG-associated diseases has lead to a nuclear toxicity model. Similar INIs are found in oculopharyngeal muscular dystrophy, which is caused by a short expansion of an alanine-encoding GCG repeat. Here we propose that transcriptional or translational frameshifts occurring within expanded CAG tracts result in the production and accumulation of polyalanine-containing mutant proteins. We hypothesize that these alanine polymers deposit in cells forming INIs and may contribute to nuclear toxicity. We show evidence that supports our hypothesis in lymphoblast cells from MJD patients, as well as in pontine neurons of MJD brain and in in vitro cell culture models of the disease. We also provide evidence that alanine polymers alone are harmful to cells and predict that a similar pathogenic mechanism may occur in the other CAG repeat disorders.

Amino Acid Sequence↗
PubMed2000

PABP2 polyalanine tract expansion causes intranuclear inclusions in oculopharyngeal muscular dystrophy.

Oculopharyngeal muscular dystrophy is caused by expansion of a (GCG)n trinucleotide repeat in the poly(A) binding protein 2 (PABP2) gene. The pathological hallmark of oculopharyngeal muscular dystrophy is the accumulation of intranuclear inclusions in muscle fibers. To test whether the polyalanine expansion of PABP2 directly leads to the formation of the nuclear aggregates, both normal and expanded PABP2 cDNAs were expressed in COS-7 cells. We find that expression of mutated PABP2 protein is sufficient for its accumulation as intranuclear inclusions.

Humans↗
PubMed1997

Detection of Activity Responsible for Induction of the Agrobacterium tumefaciens Virulence Genes in Bacteriological Agar.

Agrobacterium tumefaciens C58 grown on acidic medium containing glucose and solidified with bacteriological agar expressed a virB::lacZ fusion. No expression of this fusion was observed on a similar medium which was solidified with purified agarose. The fraction from bacteriological agar which was responsible for vir gene induction was extracted with methanol and partially purified by preparative thin-layer chromatography.

Journal Article↗
PubMed1997

Variable efficiency of a Ti plasmid-encoded VirA protein in different agrobacterial hosts.

The transconjugant CB100, harboring the Ti plasmid from the Agrobacterium tumefaciens biovar 2 strain D10B/87 in the chromosomal background of the biovar 1 strain C58, was defective in vir gene induction. This defect was corrected in the presence of virA from pTiA6. Based on this complementation result and an analysis of the induction requirements of the transconjugant CB100 and its parent strains, it was hypothesized that the defective vir gene induction in CB100 was related to a dysfunctional interaction between the pTi-encoded D10B/87 VirA and the chromosome-encoded C58 ChvE. To verify this hypothesis, D10B/87 and C58 virA were compared, and conclusions from this first set of analyses were then corroborated by comparing D10B/87 and C58 chvE. Whereas only a few nucleotide differences were identified in the promoters and 5' ends of the coding regions of D10B/87 and C58 virA, analysis of hybrid virA genes showed that these differences collectively accounted for the poor vir gene induction of strain CB100. In contrast with the sequence similarity of the VirA proteins, extensive divergence was seen between the chromosome-encoded D10B/87 and C58 ChvE. Although D10B/87 chvE introduced in trans had little effect on vir gene induction of CB100, it enhanced the induction response of a strain CB100 derivative in which the chromosomal C58 chvE had been inactivated by marker exchange. These results suggest that chromosomal backgrounds provided by different strains of A. tumefaciens are not equivalent for VirA function. Following conjugative transfer of certain Ti plasmids to a new agrobacterial host, evolution of the newly introduced virA, or coevolution of chvE and virA, may lead to optimization of ChvE-VirA interaction and vir gene induction levels.

Agrobacterium tumefaciens↗
PubMed1996

Influence of alkyltransferase activity and chromosomal locus on mutational hotspots in Chinese hamster ovary cells.

High-density mutational spectra have been established for exon 3 of the gene encoding adenine phosphoribosyltransferase (APRT) of the Chinese hamster ovary (CHO) cell line derivative D422 and closely related and/or modified lines by using the mutagen ethyl methanesulfonate (EMS). The total number of selectable sites (GC-->AT transitions yielding a selectable APRT- phenotype) was estimated at 31 based on our own accumulated data base of 136 sequenced exon 3 mutations and on literature reports. D422 and two other APRT hemizygous lines each yielded very similar spectra and showed two populations of mutable sites: (i) 24 "baseline" sites that followed the Poisson distribution and therefore were equally susceptible to mutation and (ii) two hotspots, one comprising a cluster at nucleotides 1293-1309 and the other at nucleotide 1365. Collectively, the latter sites were about 10-fold more frequently mutated than the others. CHO cells are mer- as they lack the repair enzyme O6-methylguanidine methyltransferase (EC 2.1.1.63). In modified repair-proficient CHO cells, the distribution of mutations among all of the 31 sites was random, with only 3 of the 19 GC-->AT transitions in the above hotspots. To determine whether the distribution was locus-dependent, two independent lines carrying single copies of transfected APRT genes were generated from a derivative of D422 carrying a deletion in the endogenous APRT gene. Nucleotides 1293-1309 were again no longer preferentially mutated, but the site at nucleotide 1365 was still a hotspot. We conclude that mutational spectra in mer- cells are at least in part locus dependent and that some sequences are particularly susceptible to EMS mutagenesis and perhaps also to methyltransferase repair.

Adenine Phosphoribosyltransferase↗
PubMed1995

Genetic analysis of nonpathogenic Agrobacterium tumefaciens mutants arising in crown gall tumors.

Little is known about the effect of the host on the genetic stability of bacterial plant pathogens. Crown gall, a plant disease caused by Agrobacterium tumefaciens, may represent a useful model to study this effect. Indeed, our previous observations on the natural occurrence and origin of nonpathogenic agrobacteria suggest that the host plant might induce loss of pathogenicity in populations of A. tumefaciens. Here we report that five different A. tumefaciens strains initially isolated from apple tumors produced up to 99% nonpathogenic mutants following their reintroduction into axenic apple plants. Two of these five strains were also found to produce mutants on pear and/or blackberry plants. Generally, the mutants of the apple isolate D10B/87 were altered in the tumor-inducing plasmid, harboring either deletions in this plasmid or point mutations in the regulatory virulence gene virG. Most of the mutants originating from the same tumor appeared to be of clonal origin, implying that the host plants influenced agrobacterial populations by favoring growth of nonpathogenic mutants over that of wild-type cells. This hypothesis was confirmed by coinoculation of apple rootstocks with strain D10B/87 and a nonpathogenic mutant.

Acetophenones↗
PubMed1994

Novel narrow-host-range vectors for direct cloning of foreign DNA in Pseudomonas.

Narrow-host-range vectors, based on an indigenous replicon and containing a multiple cloning site, have been constructed in a Pseudomonas host capable of growth on unusual substrates. The new cloning vectors yield sufficient amounts of DNA for preparative purposes and belong to an incompatibility group different from that of the incP and incQ broad-host-range vectors. One of these vectors, named pDB47F, was used to clone, directly in Pseudomonas, DNA fragments from Agrobacterium, Pseudomonas, and Rhizobium. A clone containing Agrobacterium and KmR gene sequences was transformed with a higher efficiency than an RSF1010-derived vector (by as much as 1250-fold) in four out of five Pseudomonas strains tested. The considerable efficiency obtained with this system makes possible the direct cloning and phenotypic selection of foreign DNA in Pseudomonas.

Base Sequence↗
PubMed1994

Effect of ionizing dose rate on the radioresistance of some food pathogenic bacteria.

Food pathogenic bacteria including Listeria monocytogenes (1A1 and ATCC 19111), Staphylococcus aureus (GD13 and ATCC 13565), Escherichia coli O157:H7 (ATCC 35150), Salmonella typhimurium, Yersinia enterocolitica, Vibrio parahaemolyticus, and Campylobacter jejuni were exposed to various rates of ionizing radiation (0.78, 2.6, and 22 kGy/h) emitted by three different 60Co irradiators. D10 values (D10 is the radiation dose required to eliminate 90% of a bacterial population (one logarithmic cycle reduction)) were calculated for the various strains and growth conditions tested. A covariance analysis of these results revealed that the dose rates studied had no significant influence on the radiosensitivity of these bacteria. At all dose rates, the bacteria were more radiosensitive when irradiated in a saline solution (0.85% NaCl) than in a chicken breast meat suspension. The growth phase of the bacterial population had a variable influence on its radioresistance. For L. monocytogenes 1A1, Staphylococcus aureus ATCC 13565, E. coli O157:H7, Y. enterocolitica, and V. parahaemolyticus, radioresistance was not significantly different in the exponential and stationary phases. Populations of L. monocytogenes ATCC 19111 and Staphylococcus aureus GD13 were significantly more resistant in the stationary phase (D10 = 0.23 and 0.12 kGy, respectively) than in the exponential phase (D10 = 0.17 and 0.09 kGy, respectively). Among the pathogenic bacteria investigated in this study, the most radioresistant was L. monocytogenes (D10 = 0.16-0.38 kGy, Gram-positive bacilli) and the most radiosensitive was V. parahaemolyticus (D10 = 0.03-0.04 kGy, halophilic Gram-negative bacilli).

Animals↗
PubMed1994

Identification of variability of ribosomal DNA spacer from Pseudomonas soil isolates.

The polymerase chain reaction was used to amplify the spacer region located between the 16S and 23S ribosomal RNA genes of strains of Pseudomonas fluorescens and Pseudomonas putida isolated from peat bog, canola field, or arctic plants. Some of spacer region of four of the P. fluorescens strains examined, strains 64-3, 63-28, QP5, and R17-FP2, was about 515 base pairs (bp) in length, and contained the genes for tRNA(Ile) and tRNA(Ala). The DNA sequences of two strains from canola, 64-3 and 63-28, differed at only two positions. The sequences of the peat bog strains QP5 and R17-FP2 were identical. However, differences were noted between the DNA sequence common to the pair of strains 64-3 and 63-28 and the corresponding common sequence for strains QP5 and R17-FP2. These differences were mainly concentrated in two DNA segments of 10 and 19 bp, respectively. A probe for the 19-bp variable segment that occurs in the ribosomal spacer of strains QP5 and R17-FP2 recognized total DNA from these two strains, but not DNA from other bacteria of different origins. These results suggest the existence of a limited degree of variability within the 16S-23S ribosomal DNA spacer region, and that this variability may be useful to the recognition of particular Pseudomonas strains from environmental samples.

Base Sequence↗
PubMed1993

Dynamic structure of Agrobacterium tumefaciens Ti plasmids.

Agrobacterium tumefaciens C58F is a variant of strain C58 which generates a high proportion of avirulent mutants in the presence of the virulence (vir) gene inducer acetosyringone. These mutants are altered in the Ti plasmid and do not respond to the acetosyringone signal (C. Fortin, E. W. Nester, and P. Dion, J. Bacteriol. 174:5676-5685, 1992). The physical organization of the Ti plasmid was compared in strain C58 and its variant. One feature distinguishing pTiC58F from its parent plasmid was the presence of the insertion element IS426. Three copies of this element were detected in the strain C58 chromosome, whereas two additional copies were found in strain C58F, including one copy in the Ti plasmid. This particular copy of IS426 was associated with the region of arginine and nopaline catabolism of pTiC58F. Most of the avirulent mutants recovered following growth of strain C58F in the presence of acetosyringone were complemented by clones carrying either virA or virG. Element IS426 was no longer found in the arginine and nopaline catabolism region of the Ti plasmids from the virA and virG mutants, but it resided in the particular KpnI fragment containing the modified vir locus. Behavior of a strain C58F derivative, which was inactivated in a chromosomal component required for the response to acetosyringone, was consistent with the possibility that vir gene induction is essential to the massive production of avirulent mutants.

Acetophenones↗
PubMed1992

Growth inhibition and loss of virulence in cultures of Agrobacterium tumefaciens treated with acetosyringone.

Acetosyringone, a phenolic inducer of the virulence (vir) genes of Agrobacterium tumefaciens, inhibited the growth of the nopaline-type strains T37 and C58 incubated under acidic conditions. In the course of a 6-day incubation with acetosyringone, avirulent clones were produced in different proportions by strains T37 and C58 and also by a spontaneous variant of strain C58, denominated C58F. The proportion of avirulent clones in acetosyringone-treated cultures often exceeded 50% for strains T37 and C58F and was of the order of 1% for strain C58. Control cultures not exposed to acetosyringone did not yield avirulent clones. Two other vir inducers, sinapinic acid and syringaldehyde, also inhibited growth and promoted accumulation of avirulent clones in cultures of strains C58F and T37. On the other hand, various acetosyringone analogs reported not to induce the vir genes did not act as growth inhibitors. All of the T37 and most of the C58F avirulent clones examined still carried a Ti plasmid. In all instances examined, avirulent clones still carrying a Ti plasmid were mutated in this plasmid. Mutants of strain C58F lacked the capacity to induce a virB::lacZ fusion in the presence of acetosyringone.

Acetophenones↗
PubMed1992

Diauxic growth of Agrobacterium tumefaciens 15955 on succinate and mannopine.

Diauxic growth was observed upon incubation of Agrobacterium tumefaciens 15955 on a mixture of succinate and mannopine as the carbon source. Diauxic growth was also observed when either fumarate or L-malate was mixed with mannopine. No diauxie was detectable when A. tumefaciens 15955 was grown on a mixture of mannopine and glucose, fructose, sucrose, or L-arabinose. Preferential utilization of succinate was observed in the initial growth phase of diauxie, whereas the final growth phase occurred at the expense of mannopine. Cells harvested during the initial growth phase exhibited a capacity for uptake of [14C]succinate but not of [14C] mannopine. A capacity for [14C]mannopine uptake was expressed during the final growth phase. Extracts from cells grown on a mixture of succinate and mannopine exhibited a low level of mannopine cyclase activity in the initial phase of diauxie. This activity increased substantially in the final phase of growth. Added succinate had no effect on the rate of [14C]mannopine uptake or mannopine cyclase activities of cells previously grown on mannopine. Diauxie was also observed during growth of strain 15955 on a mixture of succinate and octopine.

Agrobacterium tumefaciens↗
PubMed1992

Spontaneous mutation conferring the ability to catabolize mannopine in Agrobacterium tumefaciens.

Two nopaline-type strains of Agrobacterium tumefaciens, C58 and T37, as well as strain A136, which is a Ti plasmid-cured derivative of strain C58, gave rise to spontaneous mutants that were able to grow on mannopine. The observation of mutagenesis with strain A136 demonstrated that the ability to acquire this new catabolic potential was independent of the presence of a Ti plasmid. The mutants were isolated after 4 weeks of incubation on minimal medium containing mannopine as the sole carbon source. They also utilized mannopinic acid, but not agropine or agropinic acid. In addition, the spontaneous mutant LM136, but not its parent strain A136, degraded many mannityl opine analogs. [14C]mannopine disappeared in the presence of LM136 cells which had been pregrown on opine or nonopine substrates. These results suggested that the catabolic system of this mutant was not subject to a stringent regulation. A clone conferring the ability to utilize mannopine on a recipient pseudomonad was selected from a genomic library from both the mutant LM136 and its parent strain. Only the LM136 clone was expressed in the parent Agrobacterium strain A136. Southern analysis showed that the genes for mannopine catabolism in the spontaneous mutants differed from the corresponding Ti plasmid-encoded genes of octopine-type or agropine-type Agrobacterium strains. Cells of LM136 utilized [14C]mannopine without generating detectable amounts of intracellular agropine. In contrast, a major fraction of the radioactivity recovered from cells of the octopine-type strain Ach5, after incubation on [14C]mannopine, was in the form of agropine.

Agrobacterium tumefaciens↗