scholarly journals Rhizobium leguminosarum nodulation gene (nod) expression is lowered by an allele-specific mutation in the dicarboxylate transport gene dctB

Microbiology ◽  
1995 ◽  
Vol 141 (1) ◽  
pp. 103-111 ◽  
Author(s):  
A. Mavridou ◽  
M.-A. Barny ◽  
P. Poole ◽  
K. Plaskitt ◽  
A. E. Davies ◽  
...  
Keyword(s):  
Rhizobium Leguminosarum ◽  
Specific Mutation ◽  
Dicarboxylate Transport ◽  
Allele Specific

scholarly journals Transcriptomic Analysis of Rhizobium leguminosarum Biovar viciae in Symbiosis with Host Plants Pisum sativum and Vicia cracca

2009 ◽  
Vol 191 (12) ◽  
pp. 4002-4014 ◽  
Author(s):  
R. Karunakaran ◽  
V. K. Ramachandran ◽  
J. C. Seaman ◽  
A. K. East ◽  
B. Mouhsine ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Pisum Sativum ◽  
Rhizobium Leguminosarum ◽  
Tricarboxylic Acid Cycle ◽  
Dicarboxylate Transport ◽  
Magnesium Transporter ◽  
Alanine Synthesis ◽  
Vicia Cracca ◽  
Shock Proteins ◽  
Cultured Bacteria

ABSTRACT Rhizobium leguminosarum bv. viciae forms nitrogen-fixing nodules on several legumes, including pea (Pisum sativum) and vetch (Vicia cracca), and has been widely used as a model to study nodule biochemistry. To understand the complex biochemical and developmental changes undergone by R. leguminosarum bv. viciae during bacteroid development, microarray experiments were first performed with cultured bacteria grown on a variety of carbon substrates (glucose, pyruvate, succinate, inositol, acetate, and acetoacetate) and then compared to bacteroids. Bacteroid metabolism is essentially that of dicarboxylate-grown cells (i.e., induction of dicarboxylate transport, gluconeogenesis and alanine synthesis, and repression of sugar utilization). The decarboxylating arm of the tricarboxylic acid cycle is highly induced, as is γ-aminobutyrate metabolism, particularly in bacteroids from early (7-day) nodules. To investigate bacteroid development, gene expression in bacteroids was analyzed at 7, 15, and 21 days postinoculation of peas. This revealed that bacterial rRNA isolated from pea, but not vetch, is extensively processed in mature bacteroids. In early development (7 days), there were large changes in the expression of regulators, exported and cell surface molecules, multidrug exporters, and heat and cold shock proteins. fix genes were induced early but continued to increase in mature bacteroids, while nif genes were induced strongly in older bacteroids. Mutation of 37 genes that were strongly upregulated in mature bacteroids revealed that none were essential for nitrogen fixation. However, screening of 3,072 mini-Tn5 mutants on peas revealed previously uncharacterized genes essential for nitrogen fixation. These encoded a potential magnesium transporter, an AAA domain protein, and proteins involved in cytochrome synthesis.

Allele-Specific Mutation Detection

2010 ◽  
pp. 15-28
Author(s):  
Aglaia Athanassiadou ◽  
Eleana F. Stavrou ◽  
Adamandia Papachatzopoulou ◽  
George P. Patrinos
Keyword(s):  
Mutation Detection ◽  
Specific Mutation ◽  
Allele Specific

scholarly journals Inactivation and Regulation of the Aerobic C4-Dicarboxylate Transport (dctA) Gene ofEscherichia coli

1999 ◽  
Vol 181 (18) ◽  
pp. 5624-5635 ◽  
Author(s):  
Suzanne J. Davies ◽  
Paul Golby ◽  
Davood Omrani ◽  
Susan A. Broad ◽  
Vikki L. Harrington ◽  
...  
Keyword(s):  
Catabolite Repression ◽  
Sinorhizobium Meliloti ◽  
Rhizobium Leguminosarum ◽  
Northern Blot Analysis ◽  
Rhodobacter Capsulatus ◽  
Transcriptional Start Site ◽  
Receptor Protein ◽  
Growth Defect ◽  
E Coli ◽  
Dicarboxylate Transport

ABSTRACT The gene (dctA) encoding the aerobic C4-dicarboxylate transporter (DctA) of Escherichia coli was previously mapped to the 79-min region of the linkage map. The nucleotide sequence of this region reveals two candidates for the dctA gene: f428 at 79.3 min and theo157a-o424-o328 (or orfQMP) operon at 79.9 min. The f428 gene encodes a homologue of theSinorhizobium meliloti and Rhizobium leguminosarum H+/C4-dicarboxylate symporter, DctA, whereas the orfQMP operon encodes homologues of the aerobic periplasmic-binding protein- dependent C4-dicarboxylate transport system (DctQ, DctM, and DctP) ofRhodobacter capsulatus. To determine which, if either, of these loci specify the E. coli DctA system, the chromosomalf428 and orfM genes were inactivated by inserting Spr or Apr cassettes, respectively. The resulting f428 mutant was unable to grow aerobically with fumarate or malate as the sole carbon source and grew poorly with succinate. Furthermore, fumarate uptake was abolished in thef428 mutant and succinate transport was ∼10-fold lower than that of the wild type. The growth and fumarate transport deficiencies of the f428 mutant were complemented by transformation with an f428-containing plasmid. No growth defect was found for the orfM mutant. In combination, the above findings confirm that f428 corresponds to thedctA gene and indicate that the orfQMP products play no role in C4-dicarboxylate transport. Regulation studies with a dctA-lacZ (f428-lacZ) transcriptional fusion showed that dctA is subject to cyclic AMP receptor protein (CRP)-dependent catabolite repression and ArcA-mediated anaerobic repression and is weakly induced by the DcuS-DcuR system in response to C4-dicarboxylates and citrate. Interestingly, in a dctA mutant, expression ofdctA is constitutive with respect to C4-dicarboxylate induction, suggesting that DctA regulates its own synthesis. Northern blot analysis revealed a single, monocistronic dctA transcript and confirmed thatdctA is subject to regulation by catabolite repression and CRP. Reverse transcriptase-mediated primer extension indicated a single transcriptional start site centered 81 bp downstream of a strongly predicted CRP-binding site.

scholarly journals Roles of DctA and DctB in Signal Detection by the Dicarboxylic Acid Transport System of Rhizobium leguminosarum

1998 ◽  
Vol 180 (10) ◽  
pp. 2660-2669 ◽  
Author(s):  
Colm J. Reid ◽  
Philip S. Poole
Keyword(s):  
Signal Detection ◽  
Cross Talk ◽  
Rhizobium Leguminosarum ◽  
Normal Growth ◽  
Inducible Promoter ◽  
Ligand Specificity ◽  
Dicarboxylate Transport ◽  
Transposon Insertion ◽  
Gene Coding ◽  
Fusion Analysis

ABSTRACT The dctA gene, coding for the dicarboxylate transport protein, has an inducible promoter dependent on activation by the two-component sensor-regulator pair DctB and DctD. LacZ fusion analysis indicates that there is a single promoter for dctB anddctD. The dctA promoter is also induced by nitrogen limitation, an effect that requires DctB-DctD and NtrC. DctB alone is able to detect dicarboxylates in the absence of DctA and initiate transcription via DctD. However, DctA modifies signal detection by DctB such that in the absence of DctA, the ligand specificity of DctB is broader. dctAp also responds to heterologous induction by osmotic stress in the absence of DctA. This effect requires both DctB and DctD. A transposon insertion in thedctA-dctB intergenic region (dctA101) which locks transcription of dctA at a constitutive level independent of DctB-DctD results in improper signalling by DctB-DctD. Strain RU150, which carries this insertion, is defective in nitrogen fixation (Fix−) and grows very poorly on ammonia as a nitrogen source whenever the DctB-DctD signalling circuit is activated by the presence of a dicarboxylate ligand. Mutation ofdctB or dctD in strain RU150 reinstates normal growth on dicarboxylates. This suggests that DctD-P improperly regulates a heterologous nitrogen-sensing operon. Increased expression of DctA, either via a plasmid or by chromosomal duplication, restores control of DctB-DctD and allows strain RU150 to grow on ammonia in the presence of a dicarboxylate. Thus, while DctB is a sensor for dicarboxylates in its own right, it is regulated by DctA. The absence of DctA allows DctB and DctD to become promiscuous with regard to signal detection and cross talk with other operons. This indicates that DctA contributes significantly to the signalling specificity of DctB-DctD and attenuates cross talk with other operons.

Localization of the Br Polymorphism on a 144 bp Exon of the GPIa Gene and Its Application in Platelet DNA Typing

1994 ◽  
Vol 71 (05) ◽  
pp. 651-654 ◽  
Author(s):  
Rainer Kalb ◽  
Sentot Santoso ◽  
Katja Unkelbach ◽  
Volker Kiefel ◽  
Christian Mueller-Eckhardt
Keyword(s):  
Genomic Organization ◽  
Fragment Length Polymorphism ◽  
Human Platelet ◽  
Dna Typing ◽  
Rflp Analysis ◽  
Serological Typing ◽  
Allele Specific ◽  
Polymerase Chain ◽  
Alloimmune Thrombocytopenia ◽  
Rflp Typing

SummaryAlloimmunization against the human platelet alloantigen system Br (HPA-5) is the second most common cause of neonatal alloimmune thrombocytopenia (NAIT) in Caucasian populations. We have recently shown that a single base polymorphism at position 1648 on platelet mRNA coding for GPIa results in an aminoacid substitution at position 505 on the mature GPIa which is associated with the two serological defined Br phenotypes.Since DNA-typing of platelet alloantigens offers possibilities for useful clinical applications, we designed genomic DNA-based restriction fragment length polymorphism (RFLP) typing for Br alloantigens. To establish this technique we analyzed the genomic organization of GPIa adjacent to the polymorphic base. Using the polymerase chain reaction (PCR) of blood cell DNA we have identified two introns (approximately 1.7 and 1.9 kb) flanking a 144 bp coding sequence of the GPIa gene encompassing the polymorphic base 1648. Based on the in- tron sequence, a PCR primer was constructed to amplify a 274 bp fragment which was used for allele-specific RFLP to determine the Br genotypes. The results of RFLP analysis using Mnll endonuclease obtained from 15 donors (2 Br37*, 2 Br^ and 11 Brb/b) correlate perfectly with serological typing by monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay.

Simple and Rapid Detection of Factor V Leiden by Allele-specific PCR Amplification

1996 ◽  
Vol 75 (05) ◽  
pp. 757-759 ◽  
Author(s):  
Rainer Blasczyk ◽  
Markus Ritter ◽  
Christian Thiede ◽  
Jenny Wehling ◽  
Günter Hintz ◽  
...  
Keyword(s):  
Direct Sequencing ◽  
Activated Protein C ◽  
Factor V Leiden ◽  
Pcr Amplification ◽  
Factor V ◽  
Specific Pcr ◽  
Pcr Rflp ◽  
Allele Specific ◽  
Specific Amplification ◽  
Allele Specific Pcr

SummaryResistance to activated protein C is the most common hereditary cause for thrombosis and significantly linked to factor V Leiden. In this study, primers were designed to identify the factor V mutation by allele-specific PCR amplification. 126 patients with thromboembolic events were analysed using this technique, PCR-RFLP and direct sequencing. The concordance between these techniques was 100%. In 27 patients a heterozygous factor VGln506 mutation was detected, whereas one patient with recurrent thromboembolism was homozygous for the point mutation. Due to its time- and cost-saving features allele-specific amplification should be considered for screening of factor VGln506.

A Hemi-nested, Allele Specific, Whole Blood PCR Assay for the Detection of the Factor V Leiden Mutation

1997 ◽  
Vol 77 (06) ◽  
pp. 1154-1155 ◽  
Author(s):  
Gary D Sinclair ◽  
Sandra Low ◽  
Man-Chiu Poon
Keyword(s):  
Whole Blood ◽  
Protein C ◽  
Activated Protein C ◽  
Factor V Leiden ◽  
Restriction Enzyme Digestion ◽  
Factor V ◽  
Pcr Assay ◽  
Specific Primers ◽  
Factor V Leiden Mutation ◽  
Allele Specific

SummaryWe describe a novel hemi-nested, allele specific whole blood PCR assay for detection of the factor V Leiden mutation associated with the plasma defect, activated protein C resistance. This assay utilizes 5 μl of whole blood without prior DNA extraction. The hemi-nested design, employing an outer primer pair in combination with nested, allele specific primers obviates the need for restriction enzyme digestion. PCR reactions are analysed directly on agarose or polyacrylamide minigels. The assay confirmed the genotypes of 50 individuals previously categorized by PCR and Mnll digestion, and has been subsequently utilized in the genotyping of 445 individuals referred for thrombosis studies.

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