Marcadores moleculares para la taxonomía e identificación del género Brucella (Alphaproteobacteria) / Molecular markers for the taxonomy and identification of the genus Brucella (Alphaproteobacteria)

Introducción: El género Brucella está incluido en la familia Brucellaceae que pertenece al orden Rhizobiales y es reconocido por su alto grado de patogenicidad. Las bacterias de este género son responsables de la brucelosis, enfermedad que ha sido reportada como una de las zoonosis más importantes a nivel mundial por su incidencia en el ganado y el hombre. Los estudios previos para la clasificación taxonómica del género, se han basado fundamentalmente en el análisis del gen 16S ARNr. Sin embargo, pocas investigaciones se han dirigido a la identificación de marcadores moleculares que distingan a sus miembros de otros grupos de bacterias. Objetivo: Identificar inserciones en secuencias de proteínas conservadas, que pudieran ser utilizados como marcadores moleculares para la taxonomía y diagnóstico de especies del género Brucella. Métodos: Las secuencias homólogas de las proteínas analizadas fueron obtenidas de bases de datos internacionales y, posteriormente, alineadas con el programa ClustalX2, para ello fueron considerados los parámetros sugeridos en la literatura. Resultados: Se identificaron inserciones en las proteínas oxoglutarato deshidrogenasa (componente E1) y ADN ligasa A específicas del género Brucella. Conclusiones: Las inserciones halladas pueden ser empleadas como complemento a los métodos tradicionales de clasificación taxonómica y para el diagnóstico molecular de bacterias incluidas en el género Brucella(AU)

Introduction: Brucella is a genus from the Brucellaceae family, Rhizobiales order. This genus is recognized for its high pathogenicity. Brucella bacteria cause brucellosis, a disease reported as one of the most important zoonoses worldwide due to its incidence in cattle and people. Previous studies on taxonomic classification of the genus have been mainly based on the analysis of gene 16S rDNA. However, few studies have been aimed at identification of molecular markers distinguishing its members from other groups of bacteria. Objective: Identify insertions in preserved protein sequences which could be used as molecular markers for the taxonomy and diagnosis of species from the Brucella genus. Methods: The homologous sequences for the proteins analyzed were obtained from international databases and aligned with the software ClustalX2, considering the parameters suggested in the literature. Results: Insertions were identified in the proteins oxoglutarate dehydrogenase (component E1) and DNA ligase A, specific of the genus Brucella. Conclusions: The insertions found may be used as complements to the traditional methods for taxonomic classification and for the molecular diagnosis of bacteria from the genus Brucella(AU)

Biochemical and Enzymatic Characterization of a Thermostable DNA Ligase Encoded by Thermophilic Acidophilic Archaebacterium Strain JP2 / 生物化学与生物物理进展

A thermostable DNA ligase gene was identified, and the biochemical and enzymatic properties of the ligase were characterized from JP2 strain which was enriched from geothermally active sites in Papua New Guinea. The nucleotide and amino acid sequences showed much high identities compared with that of archeabacterium species Sulfolobus solfataricus and Sulfolobus shibatae,especially in the six conserved motif sequences, which are known to be closely related to the key function of ligase. Recombinant JP2 ligase showed high activity in nick-joining reaction. It was the most active when Mn2+ present as divalent metal cofactor rather than Mg2+ and Ca2+ etc.. Assay of thermostability over a range of temperatures showed that at 50～80℃ the enzyme displayed relative high activity. Further thermostability experiment indicated that the activity of JP2 ligase could last for a long time at 80℃ and 85℃,however, at 90℃ and 95℃, it became unstable quickly. An investigation on the acquired thermotolerance of recombinant JP2 ligase was done by applying a chaperonin known as TF55 in thermophile on JP2 ligase reaction. Result showed that TF55 could not help in improving thermostability of ligase at 85℃. The possible reason might be that at 85℃ in vitro, the chaperonin itself was denatured.

Relation between mutation of DNA-Ligase Ⅳ gene and radiosensitivity in human nasopharyngeal carcinoma cell lines / 中华放射肿瘤学杂志

Objective To define the correlation between mutation of DNA-LigaseⅣ gene and radiosensitivity.Methods Nasopharyngeal squamous carcinoma cell line (CNE), lung adenocarcinoma cell line (SPC-A1) and breast adenocarcinoma cell line(MCF-7) after irradiation were assessed with specific biological parameters. Polymerase chain reaction, cloning and sequencing techniques were used to determine the sequence of DNA-LigaseⅣ gene in these three cell lines. Then the impact of homologous change and mutation on hydrophicity-hydrophobicity of genic products was analyzed.Results The surviving fractions derived from irradiation were different with more radiosensitivity in the CNE cell line than in the others. In three cell lines, the homology of LigaseⅣ gene were: 99.95%,99.99%,99.98%, respectively. Some mutations including transversion and transition were detected and led to alterations in the hydrophicity-hydrophobicity function of products. Higher radiosensitivity of CNE was associated with amino-acid substitutions: 313aa His→Arg,538aa Gly→Arg,579aa Lys→Arg and 585aa Asn→Ser.Conclusion These results suggest that LigaseⅣp play an important role in the ligation of DNA double strand breaks and certain mutations bring about changes in radiosensitivity.

Cloning of the full-length rat Nor 1 cDNA using T_4 DNA-ligase-mediated 5' RACE / 中国病理生理杂志

AIM: To clone the full-length of 75A EST. METHODS: After the extraction of total RNA from primary cultured rat cerebellar granule cell of 7DIV in the medium containing 25 mmol/L KCl, T_4 DNA ligase-mediated 5' RACE was used to retrieve 5' unknown sequence of 75A EST, and the first round 5' RACE PCR product was subcloned into pGEM-T easy vector for sequence and homogeneous analysis. RESULTS: The first round of 5' RACE produce a 2.5 kb band, and 75A EST was identified to be partial sequence of Neuron-derived orphan receptor (Nor1) gene. After two more rounds RACE, we firstly cloned the full-length of Nor-1 cDNA. CONCLUSION: T_4 DNA ligase mediated 5' RACE is an efficient method to retrieve information about the 5' termini of mRNAs, and lay a foundation for further study which role Nor1 play in the cerebellar granule cell differentiation or survive.