Multiple pertussis toxin-sensitive G-proteins can couple receptors to GIRK channels in rat sympathetic neurons when expressed heterologously, but only native Gi-proteins do so in situ

Although many G-protein-coupled neurotransmitter receptors are potentially capable of modulating both voltage-dependent Ca(2+) channels (I(Ca)) and G-protein-gated K(+) channels (I(GIRK)), there is a substantial degree of selectivity in the coupling to one or other of these channels in neurons. Thus, in rat superior cervical ganglion (SCG) neurons, M(2) muscarinic acetylcholine receptors (mAChRs) selectively activate I(GIRK) whereas M(4) mAChRs selectively inhibit I(Ca). One source of selectivity might be that the two receptors couple preferentially to different G-proteins. Using antisense depletion methods, we found that M(2) mAChR-induced activation of I(GIRK) is mediated by G(i) whereas M(4) mAChR-induced inhibition of I(Ca) is mediated by G(oA). Experiments with the beta gamma-sequestering peptides alpha-transducin and beta ARK1(C-ter) indicate that, although both effects are mediated by G-protein beta gamma subunits, the endogenous subunits involved in I(GIRK) inhibition differ from those involved in I(Ca) inhibition. However, this pathway divergence does not result from any fundamental selectivity in receptor-G-protein-channel coupling because both I(GIRK) and I(Ca) modulation can be rescued by heterologously expressed G(i) or G(o) proteins after the endogenously coupled alpha-subunits have been inactivated with Pertussis toxin (PTX). We suggest instead that the divergence in the pathways activated by the endogenous mAChRs results from a differential topographical arrangement of receptor, G-protein and ion channel.     

Characterization of the termini of linear plasmids from Nectria haematococca and their use in construction of an autonomously replicating transformation vector

Summary The mitochondria of isolate FS37 from Nectria haematococca mating population I (Fusarium solani f. sp. cucurbitae) contain two linear plasmids, pFSCI and pFSC2, of 9.2 and 8.3 kbp, respectively. Evidence for a protein blocking the 5 termini of these plasmids was obtained from exonuclease digestion experiments. A single protein band with an apparent M_r of 80 K was labeled when the DNA-protein complex of either plasmid was reacted with [¹²⁵I] Bolton-Hunter reagent and then digested with DNase I. DNA sequence analysis of the termini of both plasmids revealed long inverted repeats of 1,211 by (pFSC1) and 1,027bp (pFSC2). No sequence similarity was found between the terminal inverted repeats (TIRs) of pFSC1 and pFSC2, nor was any similarity identified between the TIRs of the these plasmids and sequences of TIRs from other linear DNAs. A restriction fragment containing the TIR of pFSCI conferred autonomous replication when incorporated into an integrative transformation vector of Ustilago maydis.     

Heteroduplex analysis of Salmonella virulence plasmids and their prevalence in isolates of defined sources

Strains of the Salmonella serovars S. typhimurium, S. enteritidis, S. dublin, and S. choleraesuis harbour large plasmids which are required for extraintestinal colonization after oral infection of mice. Electron microscopic heteroduplex analysis showed that these virulence plasmids share large regions of homology. Nine hundred and eighty-six isolates of different origins were analysed for the presence of these plasmids by using a cloned fragment of a S. choleraesuis virulence plasmid as a gene probe. Virulence plasmids were detected in nearly 100% of strains isolated from animal organs or human blood. Frequencies of detection ranged from 48 to 87% in strains of faecal, food or environmental origin. These results suggest that Salmonella virulence plasmids are required for systemic infections in humans and livestock.     

Heterologous gene expression on the linear DNA killer plasmid from Kluyveromyces lactis

Summary Linear hybrid plasmids based on the killer plasmid pGKL1 from Kluyveromyces lactis were obtained by in vivo recombination in Saccharomyces cerevisiae. Like pGKL1, the hybrids are located in the cytoplasm, have terminal inverted repeats (TIR) and possess covalently linked proteins at their 5 ends. The construction of cytoplasmic hybrid plasmids is based on the use of a pGKL1 promoter to control the marker gene used for recombination. Nuclear promoters are not recognised in the cytoplasm.     

The linear mitochondrial plasmid pAL2-1 of a long-lived Podospora anserina mutant is an invertron encoding a DNA and RNA polymerase

The molecular characterization of an additional DNA species (pAL2-1) which was identified previously in a long-lived extrachromosomal mutant (AL2) of Podospora anserina revealed that this element is a mitochondrial linear plasmid. pAL2-1 is absent from the corresponding wild-type strain, has a size of 8395 bp and contains perfect long terminal inverted repeats (TIRs) of 975 bp. Exonuclease digestion experiments indicated that proteins are covalently bound at the 5 termini of the plasmid. Two long, non-overlapping open reading frames, ORF1 (3,594 bp) and ORF2 (2847 bp), have been identified, which are located on opposite strands and potentially encode a DNA and an RNA polymerase, respectively. The ORF1-encoded polypeptide contains three conserved regions which may be responsible for a 3–5 exonuclease activity and the typical consensus sequences for DNA polymerases of the D type. In addition, an amino-acid sequence motif (YSRLRT), recently shown to be conserved in terminal proteins from various bacteriophages, has been identified in the amino-terminal part of the putative protein. According to these properties, this first linear plasmid identified in P. anserina shares all characteristics with invertrons, a group of linear mobile genetic elements.     

Linear extrachromosomal DNA in the morel Morchella conica

Summary Altogether 18 different strains of the genus Morchella were assayed for the presence of extrachromosomal genetic elements. It was shown that 8 out of 13 strains of the Morchella conica group contain plasmids of comparable size (6 kb and 8 kb respectively). The 5 representatives of Morchella esculenta were not found to contain extrachromosomal DNA. The plasmid of one strain (nr. 3) was further analysed. By restriction analyses and electron microscopy it was confirmed that the plasmid is linear having a molecular weight of 6 kb. It was further shown that it carries at both ends inverted repeats of 0.75 kb.     

高迁移率族蛋白1对奥沙利铂诱导的SGC-7901胃癌细胞凋亡的影响

目的 构建pDsRED2-HMGB1重组质粒,探讨转染高迁移率族蛋白1(HMGB1)基因对奥沙利铂诱导的SGC-7901细胞凋亡的影响.方法 用逆转录聚合酶链反应(RT-PCR)扩增HMGB1 mRNA全编码序列,将PCR产物插入到pDsRED2-N1载体的Xho I和EcoR I位点,构建pDsRED2-HMGB1重组质粒;通过脂质体法转染SGC-7901胃癌细胞,荧光显微镜检测HMGB1红色荧光融合蛋白表达.Western blot鉴定HMGB1蛋白表达.应用流式细胞仪(Annexin-V/PI标记)分析HMGB1过表达对奥沙利铂诱导胃癌细胞凋亡的影响.结果 成功构建真核表达质粒pDsRED2-HMGB1,转染SGC-7901细胞后,荧光显微镜下可见细胞内有HMGB1红色荧光融合蛋白的表达;流式细胞术检测发现转染pDsRED2-HMGB1后SGC-7901细胞凋亡水平较转染pDsRED2-N1组下降22.4%.结论 HMGB1过表达可抑制奥沙利铂诱导的SGC-7901胃癌细胞凋亡.     

鹅不食草水煎剂对绿脓杆菌R质粒体外消除作用的实验研究

目的 探讨中药鹅不食草水煎剂对绿脓杆菌R质粒的消除作用。方法 对临床分离的绿脓杆菌R质粒进行了检测,并选用中药鹅不食草水煎剂对该质粒进行了体外消除试验。结果 体外药物作用24h、48h、72h,R质粒消除率分别为0.7％、4.7％、46.3％,SDS对照组分别为0.3％、0.7％、1.7％。结论 鹅不食草对耐药质粒有较强的消除作用。     

人THAP11慢病毒载体的构建及表达研究

目的 构建人死亡相关蛋白11(THAP11)基因的慢病毒载体,并建立其慢病毒表达系统.方法 PCR方法获得人THAP11基因,限制性内切酶酶切和基因重组构建慢病毒载体质粒pBPLV-THAP11-myc,并通过转染HEK293细胞观察绿色荧光蛋白(GFP)的表达以及Western blot检测其表达.在脂质体介导下将重组质粒与包装质粒pLP1和pLP2、包膜质粒pLP/VSVG共转染293FT细胞包装产生慢病毒.结果 构建的质粒经PCR,酶切及测序鉴定正确;该质粒与包装质粒共转染293FT细胞获取的5.5×106TU/ml慢病毒滴度.结论 成功构建了人THAP11基因慢病毒载体质粒THAP11-myc-pBPLV,并建立了其慢病毒表达系统,为后续的应用研究奠定了基础.     

重叠重复人心钠素基因的化学合成及其克隆

根据人心钠素的氨基酸序列,借助于计算机,设计了8个寡核苷酸片段,并在DNA自动合成仪上合成了这些片段。经5′磷酸化、退火、连接等操作,这些片段组装成由两个心钠素基因首尾以终止起始码TGATG相连的串联体。将它插入质粒pRC_(23)转化E.coli TAP_(106)后,获得pY X_(40)对重组子的酶切图谱、原位杂交及DNA序列的分析表明,插入基因的方向、读框正确。