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Identification of the nicotinamide mononucleotide adenylyltransferase of Trypanosoma cruzi.

Niño CH, Forero-Baena N, Contreras LE, Sánchez-Lancheros D, Figarella K, Ramírez MH - Mem. Inst. Oswaldo Cruz (2015)

Bottom Line: In this study, a hypothetical open reading frame (ORF) for NMNAT was identified in the genome of T. cruzi.The corresponding putative protein was analysed by simulating structural models.The ORF was amplified from genomic DNA by polymerase chain reaction and was further used for the construction of a corresponding recombinant expression vector.These results comprise the first identification of an NMNAT in T. cruzi using bioinformatics and experimental tools and hence represent the first step to understanding NAD+ metabolism in these parasites.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de Investigaciones Básicas en Bioquímica, Facultad de Ciencias, Universidad Nacional de Colombia, Bogotá, Colombia.

ABSTRACT
The intracellular parasite Trypanosoma cruzi is the aetiological agent of Chagas disease, a public health concern with an increasing incidence rate. This increase is due, among other reasons, to the parasite's drug resistance mechanisms, which require nicotinamide adenine dinucleotide (NAD+). Furthermore, this molecule is involved in metabolic and intracellular signalling processes necessary for the survival of T. cruzi throughout its life cycle. NAD+biosynthesis is performed by de novo and salvage pathways, which converge on the step that is catalysed by the enzyme nicotinamide mononucleotide adenylyltransferase (NMNAT) (enzyme commission number: 2.7.7.1). The identification of the NMNAT of T. cruzi is important for the development of future therapeutic strategies to treat Chagas disease. In this study, a hypothetical open reading frame (ORF) for NMNAT was identified in the genome of T. cruzi.The corresponding putative protein was analysed by simulating structural models. The ORF was amplified from genomic DNA by polymerase chain reaction and was further used for the construction of a corresponding recombinant expression vector. The expressed recombinant protein was partially purified and its activity was evaluated using enzymatic assays. These results comprise the first identification of an NMNAT in T. cruzi using bioinformatics and experimental tools and hence represent the first step to understanding NAD+ metabolism in these parasites.

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Related in: MedlinePlus

open reading frame amplification of nicotinamide mononucleotideadenylyltransferase of Trypanosoma cruzi from genomic DNA.Electrophoresis on a 1% agarose gel in Tris/borate/ethylenediamine tetraaceticacid of the amplified fragment. Lanes correspond to molecular marker (100 bpladder), amplicons from genomic DNA using annealing temperatures of 50°C and52ºC, followed by the negative control (polymerase chain reaction with notemplate). For subsequent experiments, a temperature of 50ºC was used.
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f03: open reading frame amplification of nicotinamide mononucleotideadenylyltransferase of Trypanosoma cruzi from genomic DNA.Electrophoresis on a 1% agarose gel in Tris/borate/ethylenediamine tetraaceticacid of the amplified fragment. Lanes correspond to molecular marker (100 bpladder), amplicons from genomic DNA using annealing temperatures of 50°C and52ºC, followed by the negative control (polymerase chain reaction with notemplate). For subsequent experiments, a temperature of 50ºC was used.

Mentions: TcNMNAT cloning - Taking into account the sequence oftheTcCLB.507047.170 gene from the genome of CL BrenerEsmeraldo-like, primers for the subsequent PCR amplification were designed.T.cruzi strain CL Brener genomic DNA was used as a template. A single fragmentwith an approximate size of 870 bp was amplified according to the electrophoresisanalysis (Fig. 3). Two annealing temperaturesbelow the hypothetical value for the primers were used to observe which condition wouldrender more specific amplification. This fragment was directly ligated into theexpression vector pET100 D-TOPO, followed by transformation into the maintenanceE. coli strain TOP10.


Identification of the nicotinamide mononucleotide adenylyltransferase of Trypanosoma cruzi.

Niño CH, Forero-Baena N, Contreras LE, Sánchez-Lancheros D, Figarella K, Ramírez MH - Mem. Inst. Oswaldo Cruz (2015)

open reading frame amplification of nicotinamide mononucleotideadenylyltransferase of Trypanosoma cruzi from genomic DNA.Electrophoresis on a 1% agarose gel in Tris/borate/ethylenediamine tetraaceticacid of the amplified fragment. Lanes correspond to molecular marker (100 bpladder), amplicons from genomic DNA using annealing temperatures of 50°C and52ºC, followed by the negative control (polymerase chain reaction with notemplate). For subsequent experiments, a temperature of 50ºC was used.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4660618&req=5

f03: open reading frame amplification of nicotinamide mononucleotideadenylyltransferase of Trypanosoma cruzi from genomic DNA.Electrophoresis on a 1% agarose gel in Tris/borate/ethylenediamine tetraaceticacid of the amplified fragment. Lanes correspond to molecular marker (100 bpladder), amplicons from genomic DNA using annealing temperatures of 50°C and52ºC, followed by the negative control (polymerase chain reaction with notemplate). For subsequent experiments, a temperature of 50ºC was used.
Mentions: TcNMNAT cloning - Taking into account the sequence oftheTcCLB.507047.170 gene from the genome of CL BrenerEsmeraldo-like, primers for the subsequent PCR amplification were designed.T.cruzi strain CL Brener genomic DNA was used as a template. A single fragmentwith an approximate size of 870 bp was amplified according to the electrophoresisanalysis (Fig. 3). Two annealing temperaturesbelow the hypothetical value for the primers were used to observe which condition wouldrender more specific amplification. This fragment was directly ligated into theexpression vector pET100 D-TOPO, followed by transformation into the maintenanceE. coli strain TOP10.

Bottom Line: In this study, a hypothetical open reading frame (ORF) for NMNAT was identified in the genome of T. cruzi.The corresponding putative protein was analysed by simulating structural models.The ORF was amplified from genomic DNA by polymerase chain reaction and was further used for the construction of a corresponding recombinant expression vector.These results comprise the first identification of an NMNAT in T. cruzi using bioinformatics and experimental tools and hence represent the first step to understanding NAD+ metabolism in these parasites.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de Investigaciones Básicas en Bioquímica, Facultad de Ciencias, Universidad Nacional de Colombia, Bogotá, Colombia.

ABSTRACT
The intracellular parasite Trypanosoma cruzi is the aetiological agent of Chagas disease, a public health concern with an increasing incidence rate. This increase is due, among other reasons, to the parasite's drug resistance mechanisms, which require nicotinamide adenine dinucleotide (NAD+). Furthermore, this molecule is involved in metabolic and intracellular signalling processes necessary for the survival of T. cruzi throughout its life cycle. NAD+biosynthesis is performed by de novo and salvage pathways, which converge on the step that is catalysed by the enzyme nicotinamide mononucleotide adenylyltransferase (NMNAT) (enzyme commission number: 2.7.7.1). The identification of the NMNAT of T. cruzi is important for the development of future therapeutic strategies to treat Chagas disease. In this study, a hypothetical open reading frame (ORF) for NMNAT was identified in the genome of T. cruzi.The corresponding putative protein was analysed by simulating structural models. The ORF was amplified from genomic DNA by polymerase chain reaction and was further used for the construction of a corresponding recombinant expression vector. The expressed recombinant protein was partially purified and its activity was evaluated using enzymatic assays. These results comprise the first identification of an NMNAT in T. cruzi using bioinformatics and experimental tools and hence represent the first step to understanding NAD+ metabolism in these parasites.

Show MeSH
Related in: MedlinePlus