转谷氨酰胺酶2抑制H1亚型流感病毒在MDCK细胞中的增殖

组织转谷酰胺酶(transglutaminase 2,TGM2)是一种普遍存在的多功能蛋白,与不同细胞的粘附和肿瘤形成有关.有证据表明,TGM2参与了宿主细胞与病毒间的相互作用,但是对于流感病毒在细胞内增殖的影响还未有报道.为了探究MDCK细胞中TGM2对H1N1亚型流感病毒增殖的影响,本研究构建了TGM2过表达和敲除...     

基于RNA-seq技术对乌头属铁棒锤中自交不亲和S基因的挖掘与分析

以毛茛科乌头属铁棒锤 (Aconitum pendulum N. Busch)2个品系‘蓝花铁棒锤’(‘WSYB1’)和‘黄花铁棒锤’(‘WSYY1’)为材料, 对其进行转录组测序(RNA-seq), 采用生物信息学方法鉴定其中可能存在的花柱S基因(self-incompatibility gene)和花粉S基因, 并...     

Genome‐scale screening in a rat haploid system identifies Thop1 as a modulator of pluripotency exit

ObjectivesThe rats are crucial animal models for the basic medical researches. Rat embryonic stem cells (ESCs), which are widely studied, can self‐renew and exhibit pluripotency in long‐term culture, but the mechanism underlying how they exit pluripotency remains obscure. To investigate the key modulators on pluripotency exiting in rat ESCs, we perform genome‐wide screening using a unique rat haploid system.Materials and MethodsRat haploid ESCs (haESCs) enable advances in the discovery of unknown functional genes owing to their homozygous and pluripotent characteristics. REX1 is a sensitive marker for the naïve pluripotency that is often utilized to monitor pluripotency exit, thus rat haESCs carrying a Rex1‐GFP reporter are used for genetic screening. Genome‐wide mutations are introduced into the genomes of rat Rex1‐GFP haESCs via piggyBac transposon, and differentiation‐retarded mutants are obtained after random differentiation selection. The exact mutations are elucidated by high‐throughput sequencing and bioinformatic analysis. The role of candidate mutation is validated in rat ESCs by knockout and overexpression experiments, and the phosphorylation of ERK1/2 (p‐ERK1/2) is determined by western blotting.ResultsHigh‐throughput sequencing analysis reveals numerous insertions related to various pathways affecting random differentiation. Thereafter, deletion of Thop1 (one candidate gene in the screened list) arrests the differentiation of rat ESCs by inhibiting the p‐ERK1/2, whereas overexpression of Thop1 promotes rat ESCs to exit from pluripotency.ConclusionsOur findings provide an ideal tool to study functional genomics in rats: a homozygous haploid system carrying a pluripotency reporter that facilitates robust discovery of the mechanisms involved in the self‐renewal or pluripotency of rat ESCs.

Differentiation of pluripotent rat embryonic stem cells (ESCs) in vitro is difficult to achieve for unknown mechanisms. Rat haploid ESCs (haESCs) have been validated as a powerful tool to target unknown functional genes and pathways based on homozygous genetic screening. Xu et al. utilized Rex1‐GFP labelled‐rat haESCs to conduct genome‐scale screening of genes modulating pluripotency exiting. Validation experiments showed that Thop1 (one of the screened out genes) played very important roles in the random differentiation of rat ESCs in vitro via modulating phosphorylation of ERK.     

Identification of phosphorylation sites in the PKD1-encoded protein C-terminal domain.

The PKD1-encoded protein, "polycystin-1", has a large N-terminal extracellular portion, multiple transmembrane domains, and a short intracellular C-terminal tail with four tyrosine residues and two putative sites for serine phosphorylation. Its function in kidney development and autosomal dominant polycystic kidney disease (ADPKD) is still unknown. We have subcloned the cDNA encoding the polycystin-1 C-terminal domain (PKD1-CTD) into a prokaryotic expression vector, and site-directed mutagenesis was performed to target the four tyrosine residues and four serine residues in two putative phosphorylation sites. In vitro phosphorylation assays were conducted on both wild type and mutant PKD1-CTD fusion proteins. It was found that the wild type PKD1-CTD and all mutant fusion proteins, except S4251G/S4252G, could be phosphorylated by lysates from cultured normal human renal collecting tubule (NHCT) cells, as well as by commercially purified cAMP-dependent protein kinase (PKA). The phosphorylation of the PKD1-CTD fusion protein by NHCT lysates was greatly enhanced by cAMP and its analog 8-Br-cAMP, and inhibited by the specific PKA inhibitors PKI(6-22) and H-89. Activators and inhibitors of protein kinase C (PKC) had no effects on the phosphorylation of the PKD1-CTD fusion protein. Using commercially purified pp60(c-src) (c-src) it was also shown that the PKD1-CTD fusion protein could be phosphorylated by c-src in vitro, and that this phosphorylation could be abolished by a mutation Y4237F. By comparing the amino acid sequence at 4249-4253 (RRSSR) with the consensus sequence for PKA phosphorylation (RRXSX), we suggest that the serine residue at 4252 is the target of phosphorylation by a cAMP-dependent protein kinase in NHCT cell lysates. In addition, we suggest that Y4237 might be phosphorylated by c-src in living cells.     

Cyanobacterial phytochrome-like PixJ1 holoprotein shows novel reversible photoconversion between blue- and green-absorbing forms

The gene, pixJ1 (formerly pisJ1), is predicted to encode a phytochrome-like photoreceptor that is essential for positive phototaxis in the unicellular cyanobacterium Synechocystis sp. PCC 6803 [Yoshihara et al. (2000) Plant Cell Physiol. 41: 1299]. The PixJ1 protein was overexpressed as a fusion with a poly-histidine tag (His-PixJ1) and isolated from Synechocystis cells. A zinc-fluorescence assay suggested that a linear tetrapyrrole was covalently attached to the His-PixJ1 protein as a chromophore. His-PixJ1 showed novel photoreversible conversion between a blue light-absorbing form (Pb, lambdaAmax=425-435 nm) and a green light-absorbing form (Pg, lambdaAmax=535 nm). Dark incubation led Pg to revert to Pb, indicative of stability of the Pb form in darkness. Red or far-red light irradiation, which is effective for photochemical conversion of the known phytochromes, produced no change in the spectra of Pb and Pg forms. Site-directed mutagenesis revealed that a Cys-His motif in the second GAF domain of PixJ1 is responsible for binding of the chromophore. Possible chromophore species are discussed with regard to the novel photoconversion spectrum.