Since its discovery a decade ago, microRNA has been identified as one of the major regulatory gene families in eukaryotic cells. Many functions of microRNAs have been revealed both in flora and fauna in recent years, ...Since its discovery a decade ago, microRNA has been identified as one of the major regulatory gene families in eukaryotic cells. Many functions of microRNAs have been revealed both in flora and fauna in recent years, but the transcriptional regulation of microRNA genes is not well-understood. In the present study, a series of primers were designed in the 2 000 nt upstream regions of porcine miR-181 and miR-1 and then the sequences were cloned into pGL3-basic vector to test their transcriptional activity. Dual-luciferase reporter assays showed that, the activity of 5＂-flanking sequence of miR-181 started on construct -51, decreasing with the length of the fragment up to -444. The upstream 590 bp confer maximal transcriptional activity and the basal promoter activity is located within the -82 to ＋16 bp region. For miR-1, the activity starts on construct -50, decreasing with the length of the fragment up to -1 254 in despite of a bit of fluctuation, and the basal promoter activity is located within the -50 to ＋47 bp region. Furthermore, some putative regulation elements of both miR-181 and miR-1 were located. In addition, tissue distribution revealed that miR-181 is expressed at a relatively low level.展开更多
The lincosamide class of antibacterials is widely used for the treatment of a broad spectrum of infections, and one prevalent route of resistance to lincosamides in pathogenic gram-positive cocco is antibiotic modific...The lincosamide class of antibacterials is widely used for the treatment of a broad spectrum of infections, and one prevalent route of resistance to lincosamides in pathogenic gram-positive cocco is antibiotic modification. Enzymes encoded by lin genes, belonging to nucleotidyltransferase superfamily, catalyze adenylylation to inactivate lincosamides. LinA can adenylylate lincosamides at either 3?-or 4?-OH of the methylthiolincosamide sugar. The crystal structure of LinA/lincomycin has confirmed its active site. However, the residue interacting with nucleotidyl donors remains elusive. Here, we modeled the complex structure of LinA/lincomycin/Mg^(2+)/AMPCPP to reveal a putative pocket for nucleotidyl donors and suggested the residue R45 in this pocket involved in the recognition of donor substrates NTP and catalysis. ITC and enzyme activity assays show that the mutation of residue R45 impairs LinA nucleotidyltransferase activity in vitro. This work provides insights into the molecular mechanism of the nucleotide binding and transferring activity of antibiotic NTases.展开更多
基金funded by the National Natural Science Foundation of China (31000996)the International Foundation for Science (B/4534-1)the Specialized Research Fund for the Doctoral Program of Higher Education, China(200805041012)
文摘Since its discovery a decade ago, microRNA has been identified as one of the major regulatory gene families in eukaryotic cells. Many functions of microRNAs have been revealed both in flora and fauna in recent years, but the transcriptional regulation of microRNA genes is not well-understood. In the present study, a series of primers were designed in the 2 000 nt upstream regions of porcine miR-181 and miR-1 and then the sequences were cloned into pGL3-basic vector to test their transcriptional activity. Dual-luciferase reporter assays showed that, the activity of 5＂-flanking sequence of miR-181 started on construct -51, decreasing with the length of the fragment up to -444. The upstream 590 bp confer maximal transcriptional activity and the basal promoter activity is located within the -82 to ＋16 bp region. For miR-1, the activity starts on construct -50, decreasing with the length of the fragment up to -1 254 in despite of a bit of fluctuation, and the basal promoter activity is located within the -50 to ＋47 bp region. Furthermore, some putative regulation elements of both miR-181 and miR-1 were located. In addition, tissue distribution revealed that miR-181 is expressed at a relatively low level.
基金supported by the National Natural Science Foundation of China(31470741)Natural Science Foundation of Fujian Province(2016J01173)+3 种基金the Key Project of Fujian Province(2017N0031)the STS project of Chinese Academy of Sciences and Fujian Province(2016T3041)the medical talent training project of Fujian provincial health and family planning commission(2016-ZQN-19)National Thousand Talents Program of China
文摘The lincosamide class of antibacterials is widely used for the treatment of a broad spectrum of infections, and one prevalent route of resistance to lincosamides in pathogenic gram-positive cocco is antibiotic modification. Enzymes encoded by lin genes, belonging to nucleotidyltransferase superfamily, catalyze adenylylation to inactivate lincosamides. LinA can adenylylate lincosamides at either 3?-or 4?-OH of the methylthiolincosamide sugar. The crystal structure of LinA/lincomycin has confirmed its active site. However, the residue interacting with nucleotidyl donors remains elusive. Here, we modeled the complex structure of LinA/lincomycin/Mg^(2+)/AMPCPP to reveal a putative pocket for nucleotidyl donors and suggested the residue R45 in this pocket involved in the recognition of donor substrates NTP and catalysis. ITC and enzyme activity assays show that the mutation of residue R45 impairs LinA nucleotidyltransferase activity in vitro. This work provides insights into the molecular mechanism of the nucleotide binding and transferring activity of antibiotic NTases.
