鸡miR-17-92基因簇上游A/T富集区启动子活性鉴定与分析

文章采用基因定点突变和报告基因技术作A/T富集区启动子鉴定和转录调控分析,研究鸡miR-17-92基因簇上游A/T富集区是否具有启动子活性。PromPredict预测分析提示miR-17-92基因簇上游-388～-444 bp处可能是启动子区域。转录因子结合位点分析发现,A/T富集区存在3个E2F1潜在结合位点,分别位于miR-17-92基因簇上游-1 273 bp(结合位点1)、-1 186 bp(结合位点2)和-753 bp(结合位点3)处。报告基因活性分析发现,鸡miR-17-92基因簇上游A/T富集区具有启动子活性,其中miR-17-92基因簇上游-440/-1区域启动子活性最强。共转染分析显示,转录因子E2F1极显著抑制该A/T富集区启动子活性(P<0.01);进一步定点突变分析表明E2F1通过E2F1结合位点1和2抑制A/T富集区启动子活性。报告基因分析发现,与对A/T富集区启动子作用不同,E2F1促进miR-17-92基因簇宿主基因(MIR17HG)启动子活性。文章首次发现鸡miR-17-92基因簇上游存在一个新的转录调控区,对揭示鸡miR-17-92基因簇转录调控具有重要意义。     

A monoclonal antibody to the alpha subunit of Gk blocks muscarinic activation of atrial K+ channels

The activated heterotrimeric guanine nucleotide binding (G) protein Gk, at subpicomolar concentrations, mimics muscarinic stimulation of a specific atrial potassium current. Reconstitution studies have implicated the alpha and beta gamma subunits as mediators, but subunit coupling by the endogenous G protein has not been analyzed. To study this process, a monoclonal antibody (4A) that binds to alpha k but not to beta gamma was applied to the solution bathing an inside-out patch of atrial membrane; the antibody blocked carbachol-activated currents irreversibly. The state of the endogenous Gk determined its susceptibility to block by the antibody. When agonist was absent or when activation by muscarinic stimulation was interrupted by withdrawal of guanosine triphosphate (GTP) in the presence or absence of guanosine diphosphate (GDP), the effects of the antibody did not persist. Thus, monoclonal antibody 4A blocked muscarinic activation of potassium channels by binding to the activated G protein in its holomeric form or by binding to the dissociated alpha subunit.     

小麦TaAGPL1基因上游转录因子TabHLH39的分离及其功能研究

腺苷二磷酸葡萄糖焦磷酸化酶 (ADP-glucose pyrophosphorylase, AGPase) 是作物淀粉合成的关键酶，由两个小亚基 (small subunit, AGPS) 和两个大亚基 (large subunit, AGPL)组成，具有细胞质 (cytosol) 和质体 (plastid) 两种亚型。AGPL1是胞质型大亚基，对该酶活性的发挥具有重要功能。已有研究表明，过表达小麦胞质AGPase大亚基TaAGPL1-1D基因显著提高了小麦AGPase活性和淀粉积累速率，说明TaAGPL1-1D在淀粉生物合成中起着重要作用。将TaAGPL1-1D启动子与小麦籽粒cDNA文库进行酵母单杂交，筛选出一个转录因子TabHLH39，随后对TabHLH39与TaAGPL1-1D启动子之间的互作进行了验证；采用大麦条纹花叶病毒诱导的基因沉默技术，在田间小麦植株基因组内沉默了该转录因子，发现 BSMV-VIGS-TabHLH39 病毒沉默小麦植株籽粒的粒长、粒宽、粒重和淀粉含量均显著下降，且TaAGPL1-1D的表达水平也显著降低，表明TabHLH39可能通过正向调控TaAGPL1-1D基因的表达，参与了小麦淀粉的合成。     

小麦TaAGPL1基因上游转录因子TabHLH39的分离及其功能研究

腺苷二磷酸葡萄糖焦磷酸化酶 (ADP-glucose pyrophosphorylase, AGPase) 是作物淀粉合成的关键酶，由两个小亚基 (small subunit, AGPS) 和两个大亚基 (large subunit, AGPL)组成，具有细胞质 (cytosol) 和质体 (plastid) 两种亚型。AGPL1是胞质型大亚基，对该酶活性的发挥具有重要功能。已有研究表明，过表达小麦胞质AGPase大亚基TaAGPL1-1D基因显著提高了小麦AGPase活性和淀粉积累速率，说明TaAGPL1-1D在淀粉生物合成中起着重要作用。将TaAGPL1-1D启动子与小麦籽粒cDNA文库进行酵母单杂交，筛选出一个转录因子TabHLH39，随后对TabHLH39与TaAGPL1-1D启动子之间的互作进行了验证；采用大麦条纹花叶病毒诱导的基因沉默技术，在田间小麦植株基因组内沉默了该转录因子，发现 BSMV-VIGS-TabHLH39 病毒沉默小麦植株籽粒的粒长、粒宽、粒重和淀粉含量均显著下降，且TaAGPL1-1D的表达水平也显著降低，表明TabHLH39可能通过正向调控TaAGPL1-1D基因的表达，参与了小麦淀粉的合成。     

Sequence and expression of mRNAs encoding the alpha 1 and alpha 2 subunits of a DHP-sensitive calcium channel

Complementary DNAs were isolated and used to deduce the primary structures of the alpha 1 and alpha 2 subunits of the dihydropyridine-sensitive, voltage-dependent calcium channel from rabbit skeletal muscle. The alpha 1 subunit, which contains putative binding sites for calcium antagonists, is a hydrophobic protein with a sequence that is consistent with multiple transmembrane domains and shows structural and sequence homology with other voltage-dependent ion channels. In contrast, the alpha 2 subunit is a hydrophilic protein without homology to other known protein sequences. Nucleic acid hybridization studies suggest that the alpha 1 and alpha 2 subunit mRNAs are expressed differentially in a tissue-specific manner and that there is a family of genes encoding additional calcium channel subtypes.     

Prostaglandin E2 promotes colon cancer cell growth through a Gs-axin-beta-catenin signaling axis

How cyclooxygenase-2 (COX-2) and its proinflammatory metabolite prostaglandin E2 (PGE2) enhance colon cancer progression remains poorly understood. We show that PGE2 stimulates colon cancer cell growth through its heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptor, EP2, by a signaling route that involves the activation of phosphoinositide 3-kinase and the protein kinase Akt by free G protein betagamma subunits and the direct association of the G protein alphas subunit with the regulator of G protein signaling (RGS) domain of axin. This leads to the inactivation and release of glycogen synthase kinase 3beta from its complex with axin, thereby relieving the inhibitory phosphorylation of beta-catenin and activating its signaling pathway. These findings may provide a molecular framework for the future evaluation of chemopreventive strategies for colorectal cancer.     

Expression of high-affinity binding of human immunoglobulin E by transfected cells

The receptor with high affinity for immunoglobulin E (IgE) on mast cells and basophils is critical in initiating allergic reactions. It is composed of an IgE-binding alpha subunit, a beta subunit, and two gamma subunits. The human alpha subunit was expressed on transfected cells in the presence of rat beta and gamma subunits or in the presence of the gamma subunit alone. The IgE binding properties of the expressed human alpha were characteristic of receptors on normal human cells. These results now permit a systematic analysis of human IgE binding and a search for therapeutically useful inhibitors of that binding.     

Selective inhibition of a regulatory subunit of protein phosphatase 1 restores proteostasis

Many biological processes are regulated through the selective dephosphorylation of proteins. Protein serine-threonine phosphatases are assembled from catalytic subunits bound to diverse regulatory subunits that provide substrate specificity and subcellular localization. We describe a small molecule, guanabenz, that bound to a regulatory subunit of protein phosphatase 1, PPP1R15A/GADD34, selectively disrupting the stress-induced dephosphorylation of the α subunit of translation initiation factor 2 (eIF2α). Without affecting the related PPP1R15B-phosphatase complex and constitutive protein synthesis, guanabenz prolonged eIF2α phosphorylation in human stressed cells, adjusting the protein production rates to levels manageable by available chaperones. This favored protein folding and thereby rescued cells from protein misfolding stress. Thus, regulatory subunits of phosphatases are drug targets, a property used here to restore proteostasis in stressed cells.     

鸭IL-2基因启动子的克隆、多态性与表达的关联分析

为研究鸭IL-2基因调控区单核苷酸多态性对其转录调控的影响,克隆获得了鸭IL-2基因启动子2 850 bp序列,与人、小鼠和原鸡的同源性分别为35.37%,37.52%和34.74%。其中,-1 400/-1 000存在集中的核心转录因子结合位点。对鸭IL-2基因启动子(-1 932/-742)进行单核苷酸多态检测和遗传多态性分析发现,该区域存在两个突变位点(C-1353A、C-1406T),且均处于Hardy-Weinberg极不平衡状态;等位基因A均为优势等位基因。单核苷酸多态与其表达水平的相关性分析发现,突变位点不同基因型与IL-2基因mRNA表达水平和IL-2蛋白水平均无显著相关关系,但基因型AA个体的mRNA表达量均高于其他基因型个体(P>0.05),表明鸭IL-2启动子等位基因A可能有促进IL-2基因转录的趋势。研究结果为IL-2基因转录调控机制的研究提供了理论基础。     

Isolation and Sequence Analysis of HMW Glutenin Subunit 1Dy10.1 Ecoding Gene from Xinjiang Wheat (Triticum petropavlovskyi Udacz.et Migusch)

A novel HMW glutenin subunit gene 1Dy10.1 was isolated and characterized from Xinjiang wheat (Triticum petropavlovskyi. Udacz. et Migusch) accession Daomai 2. The complete open reading frame (ORF) of 1Dy10.1 was 1965 bp, encoding 655 amino acids. The numbers and distribution of cysteines in 1Dy10.1 were similar to those of 1Dy10 and other y-type subunits. In the N-terminal of 1Dy10.1, an amino acid was changed from L (leucine) to P (proline) at position 55. The repetitive domain of 1Dy10.1 differed from those of known HMW subunits by substitutions, insertions or/and deletions involving single or more amino acid residues. In the repetitive domain of subunit 1Dy10.1, the deletion of tripeptide GQQ in the consensus unit PGQGQQ resulted in the appearance of the motif PGQ that have not been observed in other known y-type HMW subunits. In comparison with the subunit 1Dy12, a deletion of dipeptide GQ, which occurred in subunit 1Dy10, was also observed in subunit 1Dy10.1. The cloned 1Dyl0.1 gene had been successfully expressed in Escherichia coli, and the expressed protein had the identical mobility with the endogenous subunit 1Dyl0.1 from seed.     

小麦籽粒Waxy蛋白亚基动态表达规律的研究

[目的]对新疆自育的4个春小麦品种新春8号、新春9与、新春11号和新春14号的Waxy蛋白亚基在籽粒灌浆过程中的动态表达进行分析,研究扬花期后籽粒贮藏蛋白中Waxy蛋白亚基的动态累积规律,为小麦淀粉的品质改良提供理论依据.[方法]利用Waxy蛋白亚基特异分子标记,通过SDS-PAGE电泳进行Waxy蛋白亚基的检测.[结果]4个品种Waxy蛋白亚基的组成不尽相同,新春8号和新春11号Waxy蛋白3个亚基都存在,而新春9号和新春14号为Wx-B1亚基的缺失类型.新春8号亚基出现的时期较晚,花后20 d开始检测到;新春14号Wx-D1亚基的表达要晚于Wx-A1亚基;新春9号和11号亚基于花后15 d检测到,但在籽粒灌浆各时期表达强弱也不尽一致.[结论]Waxy蛋白亚基形成具有时间性,且各亚基在籽粒灌浆过程中的表达规律品种间也不尽一致.