RESUMEN
The GPAT4 gene is considered as a potential functional candidate for single nucleotide polymorphism (SNP) studies in dairy cattle breeding due to its association with dairy performance in cattle by encoding an enzyme responsible for the presence of diacylglycerols and triacylglycerols in milk. Using the example of the GPAT4 gene, we applied the minigene splicing assay to analyze the functional consequences of its variant that was predicted to affect normal splicing. The results of functional analysis revealed the sequence variations (rs442541537), transfection experiments in a wild type and mutant cell line model system demonstrated that the investigated mutation in the second intron of the GPAT4 gene was responsible for the presence of a second exon in mature messenger RNA (mRNA). The cases of its absence in the spliced mature mRNA transcript resulted in a truncated dysfunctional protein due to the appearance of a stop codon. Thus, the discovered SNP led to alternative splicing in pre-mRNA by the 'cassette exon' ('exon skipping') mechanism. The studied mutation can potentially be a molecular genetic marker for alternative splicing for the GPAT4 gene and, therefore contributes to economic benefits in cattle breeding programs.
Asunto(s)
Empalme Alternativo , Empalme del ARN , Animales , Bovinos/genética , Secuencia de Bases , Mutación/genética , Empalme Alternativo/genética , Exones/genética , ARN Mensajero/metabolismo , Empalme del ARN/genéticaRESUMEN
Tick-borne diseases cause significant livestock losses worldwide. In Russia, information concerning single or mixed infections with different Anaplasma, Theileria and Babesia species in cattle is very limited. This study was conducted to determine the level of co-infection with protozoan pathogens (Theileria spp. and Babesia spp.) and rickettsial pathogens (A. marginale and A. phagocytophilum) in cattle in central Russia. Blood samples were examined with real time polymerase chain reaction (RT-PCR) for A. marginale and A. phagocytophilum, and by amplifying the V4 hypervariable region of the 18S rRNA gene, followed by cloning, DNA sequencing, and phylogenetic analyses, for Babesia and Theileria species. In total 67% of examined blood samples were positive for Theileria spp. or A. marginale, and 19% of the animals were co-infected with Theileria spp. and A. marginale. Seasonal variation in prevalence was found for Theileria spp. Phylogenetic analysis based on 18S rRNA gene sequences revealed the presence of five Theileria species: T. annulata, T. orientalis, T. buffeli, T. sergenti, and T. sinensis. No samples were positive for Babesia spp. or A. phagocytophilum. The data obtained for prevalence of bovine theileriosis and anaplasmosis in the central part of Russia underscore the need for improved surveillance and control programs to reduce tick-borne diseases in cattle.
Asunto(s)
Anaplasma/aislamiento & purificación , Babesia/aislamiento & purificación , Enfermedades de los Bovinos/epidemiología , Coinfección/veterinaria , Theileria/aislamiento & purificación , Enfermedades por Picaduras de Garrapatas/veterinaria , Anaplasma/genética , Anaplasmosis/sangre , Anaplasmosis/epidemiología , Animales , Babesia/genética , Babesiosis/sangre , Babesiosis/epidemiología , Bovinos , Coinfección/epidemiología , ADN Protozoario/genética , Filogenia , ARN Ribosómico 18S/genética , Federación de Rusia/epidemiología , Análisis de Secuencia de ADN , Theileria/genética , Theileriosis/sangre , Theileriosis/epidemiología , Enfermedades por Picaduras de Garrapatas/epidemiologíaRESUMEN
Little is known of the bacterial communities associated with the rhizosphere of wild plant species found in natural settings. The rhizosphere bacterial community associated with wild thyme, Thymus zygis L., plants was analyzed using cultivation, the creation of a near-full length 16S rRNA gene clone library and 454 amplicon pyrosequencing. The bacterial community was dominated by Proteobacteria (mostly Alphaproteobacteria and Betaproteobacteria), Actinobacteria, Acidobacteria, and Gemmatimonadetes. Although each approach gave a different perspective of the bacterial community, all classes/subclasses detected in the clone library and the cultured bacteria could be found in the pyrosequencing datasets. However, an exception caused by inconclusive taxonomic identification as a consequence of the short read length of pyrotags together with the detection of singleton sequences which corresponded to bacterial strains cultivated from the same sample highlight limitations and considerations which should be taken into account when analysing and interpreting amplicon datasets. Amplicon pyrosequencing of replicate rhizosphere soil samples taken a year later permit the definition of the core microbiome associated with Thymus zygis plants. Abundant bacterial families and predicted functional profiles of the core microbiome suggest that the main drivers of the bacterial community in the Thymus zygis rhizosphere are related to the nutrients originating from the plant root and to their participation in biogeochemical cycles thereby creating an intricate relationship with this aromatic plant to allow for a feedback ecological benefit.
Asunto(s)
Microbiota/genética , Microbiología del Suelo , Thymus (Planta)/microbiología , Variación Genética , Tipificación Molecular , Parques Recreativos , Raíces de Plantas/microbiología , ARN Bacteriano/genética , ARN Ribosómico 16S/genética , Rizosfera , Análisis de Secuencia de ARN , EspañaRESUMEN
Adenylate kinase (AK) from the sulphate-reducing bacterium Desulfovibrio gigas (AK) has been characterized earlier as a Co(2+)/Zn(2+)-containing enzyme, which is an unusual characteristic for adenylate kinases from Gram-negative bacteria, in which these enzymes are normally devoid of metal ions. AK was overexpressed in E. coli and homogeneous Co(2+)-, Zn(2+)- and Fe(2+)-forms of the enzyme were obtained under in vivo conditions. Their structural stability and spectroscopic and kinetic properties were compared. The thermal denaturation of Co(2+)- and Zn(2+)-forms of AK was studied as a cooperative two-state process, sufficiently reversible at pH 10, which can be correctly interpreted in terms of a simple two-state thermodynamic model. In contrast, the thermally induced denaturation of Fe(2+)-AK is irreversible and strongly dependent upon the scan rate, suggesting that this process is under kinetic control. Practically identical contents of secondary-structure elements were found for all the metal-chelated-forms of AK upon analysis of circular dichroism data, while their tertiary structures were significantly different. The peculiar tertiary structure of Fe(2+)-AK, in contrast to Co(2+)- and Zn(2+)-AK, and the consequent changes in the physico-chemical and enzymatic properties of the enzyme are discussed.
Asunto(s)
Quelantes/farmacología , Cobalto/química , Desulfovibrio gigas/metabolismo , Bacterias Gramnegativas/metabolismo , Hierro/química , Zinc/química , Dicroismo Circular , Clonación Molecular , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Concentración de Iones de Hidrógeno , Cinética , Conformación Proteica , Estructura Secundaria de Proteína , Espectrofotometría/métodos , Espectrofotometría Ultravioleta/métodos , TermodinámicaRESUMEN
The structural stability of a peroxidase, a dimeric protein from palm tree Chamaerops excelsa leaves (CEP), has been characterized by high-sensitivity differential scanning calorimetry, circular dichroism and steady-state tryptophan fluorescence at pH 3. The thermally induced denaturation of CEP at this pH value is irreversible and strongly dependent upon the scan rate, suggesting that this process is under kinetic control. Moreover, thermally induced transitions at this pH value are dependent on the protein concentration, leading to the conclusion that in solution CEP behaves as dimer, which undergoes thermal denaturation coupled with dissociation. Analysis of the kinetic parameters of CEP denaturation at pH 3 was accomplished on the basis of the simple kinetic scheme N-->kD, where k is a first-order kinetic constant that changes with temperature, as given by the Arrhenius equation; N is the native state, and D is the denatured state, and thermodynamic information was obtained by extrapolation of the kinetic transition parameters to an infinite heating rate.
Asunto(s)
Arecaceae/enzimología , Peroxidasa/química , Peroxidasa/metabolismo , Temperatura , Rastreo Diferencial de Calorimetría , Dicroismo Circular , Estabilidad de Enzimas , Fluorescencia , Concentración de Iones de Hidrógeno , Peroxidasa/aislamiento & purificaciónRESUMEN
The structural stability of a peroxidase, a dimeric protein from royal palm tree (Roystonea regia) leaves, has been characterized by high-sensitivity differential scanning calorimetry, circular dichroism, steady-state tryptophan fluorescence and analytical ultracentifugation under different solvent conditions. It is shown that the thermal and chemical (using guanidine hydrochloride (Gdn-HCl)) folding/unfolding of royal palm tree peroxidase (RPTP) at pH 7 is a reversible process involving a highly cooperative transition between the folded dimer and unfolded monomers, with a free stabilization energy of about 23 kcal per mol of monomer at 25 degrees C. The structural stability of RPTP is pH-dependent. At pH 3, where ion pairs have disappeared due to protonation, the thermally induced denaturation of RPTP is irreversible and strongly dependent upon the scan rate, suggesting that this process is under kinetic control. Moreover, thermally induced transitions at this pH value are dependent on the protein concentration, allowing it to be concluded that in solution RPTP behaves as dimer, which undergoes thermal denaturation coupled with dissociation. Analysis of the kinetic parameters of RPTP denaturation at pH 3 was accomplished on the basis of the simple kinetic scheme N-->kD, where k is a first-order kinetic constant that changes with temperature, as given by the Arrhenius equation; N is the native state, and D is the denatured state, and thermodynamic information was obtained by extrapolation of the kinetic transition parameters to an infinite heating rate. Obtained in this way, the value of RPTP stability at 25 degrees C is ca. 8 kcal per mole of monomer lower than at pH 7. In all probability, this quantity reflects the contribution of ion pair interactions to the structural stability of RPTP. From a comparison of the stability of RPTP with other plant peroxidases it is proposed that one of the main factors responsible for the unusually high stability of RPTP which enhances its potential use for biotechnological purposes, is its dimerization.
Asunto(s)
Arecaceae/enzimología , Peroxidasa/química , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Pliegue de Proteína , Multimerización de Proteína , TermodinámicaRESUMEN
Native zinc/cobalt-containing ATP sulfurylase (ATPS; EC 2.7.7.4; MgATP:sulfate adenylyltransferase) from Desulfovibrio desulfuricans ATCC 27774 was purified to homogeneity and crystallized. The orthorhombic crystals diffracted to beyond 2.5 A resolution and the X-ray data collected should allow the determination of the structure of the zinc-bound form of this ATPS. Although previous biochemical studies of this protein indicated the presence of a homotrimer in solution, a dimer was found in the asymmetric unit. Elucidation of this structure will permit a better understanding of the role of the metal in the activity and stability of this family of enzymes.
Asunto(s)
Proteínas Bacterianas/química , Desulfovibrio desulfuricans/enzimología , Sulfato Adenililtransferasa/química , Sulfatos/química , Difracción de Rayos X , Proteínas Bacterianas/aislamiento & purificación , Cobalto/química , Cristalización , Activación Enzimática/fisiología , Estabilidad de Enzimas , Sulfato Adenililtransferasa/aislamiento & purificación , Zinc/químicaRESUMEN
Adenylate kinase (AK) mediates the reversible transfer of phosphate groups between the adenylate nucleotides and contributes to the maintenance of their constant cellular level, necessary for energy metabolism and nucleic acid synthesis. The AK were purified from crude extracts of two sulfate-reducing bacteria (SRB), Desulfovibrio (D.) gigas NCIB 9332 and Desulfovibrio desulfuricans ATCC 27774, and biochemically and spectroscopically characterised in the native and fully cobalt- or zinc-substituted forms. These are the first reported adenylate kinases that bind either zinc or cobalt and are related to the subgroup of metal-containing AK found, in most cases, in Gram-positive bacteria. The electronic absorption spectrum is consistent with tetrahedral coordinated cobalt, predominantly via sulfur ligands, and is supported by EPR. The involvement of three cysteines in cobalt or zinc coordination was confirmed by chemical methods. Extended X-ray absorption fine structure (EXAFS) indicate that cobalt or zinc are bound by three cysteine residues and one histidine in the metal-binding site of the "LID" domain. The sequence 129Cys-X5-His-X15-Cys-X2-Cys of the AK from D. gigas is involved in metal coordination and represents a new type of binding motif that differs from other known zinc-binding sites of AK. Cobalt and zinc play a structural role in stabilizing the LID domain.
Asunto(s)
Adenilato Quinasa/química , Cobalto/química , Desulfovibrio desulfuricans/enzimología , Desulfovibrio gigas/enzimología , Zinc/química , Absorciometría de Fotón , Adenilato Quinasa/aislamiento & purificación , Adenilato Quinasa/metabolismo , Secuencia de Aminoácidos , Apoenzimas/metabolismo , Sitios de Unión , Espectroscopía de Resonancia por Spin del Electrón , Datos de Secuencia Molecular , Peso Molecular , Concentración Osmolar , Estructura Terciaria de Proteína , Pirimidinas , Alineación de Secuencia , Espectrofotometría UltravioletaRESUMEN
Nitrate reductases are enzymes that catalyze the conversion of nitrate to nitrite. We report here electron paramagnetic resonance (EPR) studies in the periplasmic nitrate reductase isolated from the sulfate-reducing bacteria Desulfovibrio desulfuricans ATCC 27774. This protein, belonging to the dimethyl sulfoxide reductase family of mononuclear Mo-containing enzymes, comprises a single 80-kDa subunit and contains a Mo bis(molybdopterin guanosine dinucleotide) cofactor and a [4Fe-4S] cluster. EPR-monitored redox titrations, carried out with and without nitrate in the potential range from 200 to -500 mV, and EPR studies of the enzyme, in both catalytic and inhibited conditions, reveal distinct types of Mo(V) EPR-active species, which indicates that the Mo site presents high coordination flexibility. These studies show that nitrate modulates the redox properties of the Mo active site, but not those of the [4Fe-4S] center. The possible structures and the role in catalysis of the distinct Mo(V) species detected by EPR are discussed.
Asunto(s)
Proteínas Bacterianas/química , Desulfovibrio desulfuricans/enzimología , Nitrato-Reductasa/química , Proteínas Bacterianas/antagonistas & inhibidores , Sitios de Unión , Catálisis , Espectroscopía de Resonancia por Spin del Electrón , Nucleótidos de Guanina/química , Molibdeno/química , Nitrato-Reductasa/antagonistas & inhibidores , Nitratos/química , Oxidación-Reducción , Periplasma/enzimología , Pterinas/químicaRESUMEN
A novel adenylate kinase (AK) has recently been purified from Desulfovibrio gigas and characterized as a Co(2+)/Zn(2+)-containing enzyme: this is an unusual characteristic for AKs from Gram-negative bacteria, in which these enzymes are normally devoid of metals. Here, we studied the conformational stability of holo- and apo-AK as a function of temperature by differential scanning calorimetry (DSC), circular dichroism (CD), and intrinsic fluorescence spectroscopy. The thermal unfolding of AK is a cooperative two-state process, and is sufficiently reversible in the 9-11 pH range, that can be correctly interpreted in terms of a simple two-state thermodynamic model. The spectral parameters as monitored by ellipticity changes in the CD spectra of the enzyme as well as the decrease in tryptophan intensity emission upon heating were seen to be good complements to the highly sensitive but integral DSC-method.