Test systems to study transcriptional regulation and promoter activity in Bacillus megaterium

Plasmid-located (multi-copy) and chromosomally located (single-copy) promoter test systems were developed for Bacillus megaterium by making use of the homologous beta-galactosidase-encoding bgaM gene. The multi-copy system facilitates rapid promoter analyses and promoter trapping, whereas the single-copy system, integrated into the chromosome, allows investigation of tightly regulated promoters. As a prerequisite for both the multi- and the single-copy systems, a beta-galactosidase-deficient B. megaterium strain was generated by deletion mutagenesis. Both test systems were verified using the promoter of the xylose operon (P( xylA )) from B. megaterium along with its repressor (XylR). As expected, expression levels in the two systems differed significantly, although expression of the bgaM reporter gene was induced by xylose in both cases, thereby proving the functionality of both the multi- and the single-copy system.     

基因转录调控相关数据库集成系统及其应用

通过互联网访问的有关基因转录调控的数据库集成系统及其应用 ,包括调控区 (3’和 5’调控区、内显子和外显子调控区等 )、调控单元 (启动子 ,增强子 ,沉默子等 )和转录因子结合位点相关数据库及其数据库系统的性质、组成和功能。也介绍了这些数据库和系统的查询和搜索方法以及相关开发的程序工具。这些生物信息学资源对于从事生物信息学、分子生物学、遗传工程、基因功能、生物技术、代谢工程、药物设计、病理学和药理学研究的机构及人员在教学研究方面具一定的参考价值和帮助。     

Epigenetic and transcriptional regulation of autophagy

Macroautophagy (hereafter referred to as autophagy) is an essential self-digestion process to maintain homeostasis and promote survival in response to starvation. Although the components of autophagy in the cytoplasm have been well studied, little has been known about the fine-tuning mechanism of autophagy through epigenetic regulations. Recently, we identified the histone arginine methyltransferase CARM1 as a new component and followed histone H3R17 dimethylation as a critical epigenetic mark in starvation-induced autophagy. Upon nutrient starvation, CARM1 is stabilized in the nucleus, but not in the cytoplasm, whereas it is constantly degraded under nutrient-rich conditions by the SKP2-containing SCF (SKP1-CUL1-F-box protein) E3 ubiquitin ligase. We further showed that nutrient starvation induces the protein levels and activity of AMPK in the nucleus. Activated AMPK then phosphorylates FOXO3, leading to SKP2 downregulation and increased CARM1 protein levels in the nucleus. Stabilized CARM1 in turn functions as an essential co-activator of TFEB and regulates the expression of autophagy and lysosomal genes. Our findings provide a conceptual advance that activation of specific epigenetic programs is indispensable for a sustained autophagic response, and shed light on a potential therapeutic targeting of the newly identified AMPK-SKP2-CARM1 signaling axis in autophagy-related diseases.     

Southwestern blotting in investigating transcriptional regulation

Southwestern blotting is used to investigate DNA-protein interactions. The advantage of this technique over other related methods such as electrophoretic mobility shift assay (EMSA) and DNA footprinting is that it provides information regarding the molecular weight of unknown protein factor. This method combines the features of Southern and Western blotting techniques; a denaturing SDS-PAGE is first employed to separate proteins electrophoretically based on size, and after transferring the proteins to a membrane support, the membrane-bound proteins are renatured and incubated with a (32)P-labeled double-stranded oligonucleotide probe of specific DNA sequence. The interaction of the probe with the protein(s) is later visualized by autoradiography. This technique could be combined with database searching (TransFac, http://www.gene-regulation.com/pub/databases.html#transfac), prediction of potential protein factors binding onto a target motif (e.g., Patch search), in vitro supershift EMSA and in vivo chromatin immunoprecipitation (ChIP) assays for effective identification of protein factors. The whole Southwestern blotting procedure takes approximately 4 d to complete. In this article, a commonly used protocol and expected results are described and discussed.