Methanosarcina acetivorans C2A Topoisomerase IIIα, an Archaeal Enzyme with Promiscuity in Divalent Cation Dependence

Topoisomerases play a fundamental role in genome stability, DNA replication and repair. As a result, topoisomerases have served as therapeutic targets of interest in Eukarya and Bacteria, two of the three domains of life. Since members of Archaea, the third domain of life, have not been implicated in any diseased state to-date, there is a paucity of data on archaeal topoisomerases. Here we report Methanosarcina acetivorans TopoIIIα (MacTopoIIIα) as the first biochemically characterized mesophilic archaeal topoisomerase. Maximal activity for MacTopoIIIα was elicited at 30–35°C and 100 mM NaCl. As little as 10 fmol of the enzyme initiated DNA relaxation, and NaCl concentrations above 250 mM inhibited this activity. The present study also provides the first evidence that a type IA Topoisomerase has activity in the presence of all divalent cations tested (Mg2+, Ca2+, Sr2+, Ba2+, Mn2+, Fe2+, Co2+, Ni2+, Cu2+, Zn2+ and Cd2+). Activity profiles were, however, specific to each metal. Known type I (ssDNA and camptothecin) and type II (etoposide, novobiocin and nalidixic acid) inhibitors with different mechanisms of action were used to demonstrate that MacTopoIIIα is a type IA topoisomerase. Alignment of MacTopoIIIα with characterized topoisomerases identified Y317 as the putative catalytic residue, and a Y317F mutation ablated DNA relaxation activity, demonstrating that Y317 is essential for catalysis. As the role of Domain V (C-terminal domain) is unclear, MacTopoIIIα was aligned with the canonical E. coli TopoI 67 kDa fragment in order to construct an N-terminal (1–586) and a C-terminal (587–752) fragment for analysis. Activity could neither be elicited from the fragments individually nor reconstituted from a mixture of the fragments, suggesting that native folding is impaired when the two fragments are expressed separately. Evidence that each of the split domains plays a role in Zn2+ binding of the enzyme is also provided

Viabilidade dos grãos de pólen de flores de pinheira (Annona squamosa) em diferentes horários Viability of the sugar apple (Annona squamosa) pollen grains at different hours of the day

No manejo do cultivo da pinha (Annona squamosa), a polinização artificial é uma prática preconizada para, obter maior pegamento dos frutos bem como uniformização do formato dos mesmos. Nesse sentido, conduziu-se este trabalho, com o objetivo de avaliar a viabilidade dos grãos de pólen de flores de pinheira em diferentes horários de coleta. O pólen foi obtido a partir de flores no estádio funcionalmente estaminada. Foram avaliados oito horários de coleta de pólen: zero hora, 1 hora, 2 horas, 3 horas, 4 horas, 5 horas, 6 horas e 7 horas da manhã. Foi utilizado meio de cultura padrão para germinação de pólen, com concentração de 10% de sacarose. As flores foram coletadas nos horários estabelecidos e os grãos de pólen foram retirados das anteras com auxílio de um pincel número 2 e em seguida inoculados em placas de Petri contendo o meio de cultura. O delineamento experimental foi inteiramente casualizado com quatro repetições, sendo cada parcela experimental constituída por duas placas Petri. Foram contados 100 grãos de pólen por placa. Após 6 horas de inoculação, os grãos de pólen foram visualizados sob lupa. Foram considerados germinados os grãos de pólen que possuíam tubo polínico com tamanho igual ou superior ao diâmetro do próprio pólen. Não foram observadas diferenças significativas entre o horário de coleta dos grãos de pólen. A percentagem média da germinação variou de 46,75% a 53,62% dos grãos de pólen germinados.<br>In the management of the sugar apple (Annona squamosa) crop, the artificial pollination is a preconized practice to obtain higher establishment of the fruits as well as their standardization. This study was carried out to evaluate the viability of sugar apple pollen grains at different collecting times. The pollens were obtained from the flowers at the functional staminate stage. The pollen grains were collected every hour, starting from 00:00am and ending at 07:00am, totalizing 8 collections. The standard culture medium with 10% sucrose was used for the germination of the pollen. The flowers were collected at the scheduled hours and the pollen grains were taken from the anthers, by using a brush # 2. They were then inoculated on Petri dishes containing the culture medium. The completely randomized experimental design was used with four replicates, and each plot was constituted by two Petri dishes. One hundred pollen grains were counted in each Petri dish. After six hours of inoculation, the pollen grains were visualized under magnifying glass. Those pollen grains containing the pollinic tube with the same or higher size than their own pollen diameter were considered germinated. No significant differences were found among the different hours of the pollen grain collections, and the germination percentage ranged from 46.75% to 53.62%