Sequence and structure of a human glucose transporter

The amino acid sequence of the glucose transport protein from human HepG2 hepatoma cells was deduced from analysis of a complementary DNA clone. Structural analysis of the purified human erythrocyte glucose transporter by fast atom bombardment mapping and gas phase Edman degradation confirmed the identity of the clone and demonstrated that the HepG2 and erythrocyte transporters are highly homologous and may be identical. The protein lacks a cleavable amino-terminal signal sequence. Analysis of the primary structure suggests the presence of 12 membrane-spanning domains. Several of these may form amphipathic alpha helices and contain abundant hydroxyl and amide side chains that could participate in glucose binding or line a transmembrane pore through which the sugar moves. The amino terminus, carboxyl terminus, and a highly hydrophilic domain in the center of the protein are all predicted to lie on the cytoplasmic face. Messenger RNA species homologous to HepG2 glucose transporter messenger RNA were detected in K562 leukemic cells, HT29 colon adenocarcinoma cells, and human kidney tissue.     

稳定表达T_7RNA聚合酶的猪睾丸细胞系的建立

根据GenBank中登录的T7RNA聚合酶基因参考序列,设计合成了1对特异性引物扩增对T7RNA聚合酶基因进行扩增,将测序正确的T7RNA聚合酶基因和真核表达载体pIRES2-EGFP双酶切后进行连接构建pIRES2-EGFP-T7RNA RNA质粒。再将构建正确的pIRES2-EGFP-T7RNA质粒经用脂质体法转染猪睾丸细胞,通过G418筛选和单细胞克隆化,同时构建pET-32a-RED原核表达质粒载体,用其检测T7启动子控制下的红色荧光蛋白的表达。结果表明,建立的ST/T7RNA细胞系经20次传代仍然能稳定表达T7RNA聚合酶。结果显示,成功建立能稳定表达T7RNA聚合酶的猪睾丸细胞系,为猪瘟病毒反向遗传操作平台奠定了基础。     

Structure of a transcribing T7 RNA polymerase initiation complex

The structure of a T7 RNA polymerase (T7 RNAP) initiation complex captured transcribing a trinucleotide of RNA from a 17-base pair promoter DNA containing a 5-nucleotide single-strand template extension was determined at a resolution of 2.4 angstroms. Binding of the upstream duplex portion of the promoter occurs in the same manner as that in the open promoter complex, but the single-stranded template is repositioned to place the +4 base at the catalytic active site. Thus, synthesis of RNA in the initiation phase leads to accumulation or "scrunching" of the template in the enclosed active site pocket of T7 RNAP. Only three base pairs of heteroduplex are formed before the RNA peels off the template.