Werner syndrome protein: functions in the response to DNA damage and replication stress in S-phase

Werner syndrome (WS) is an excellent model system for the study of human aging. WRN, a nuclear protein mutated in WS, plays multiple roles in DNA metabolism. Our understanding about the metabolic regulation and function of this RecQ helicase has advanced greatly during the past decade, largely due to the availability of purified WRN protein, WRN knockdown cells, and WRN knockout mice. Recent biochemical and genetic studies indicate that WRN plays significant roles in DNA replication, DNA repair, and telomere maintenance. Interestingly, many WRN functions require handling of DNA ends during S-phase, and evidence suggests that WRN plays both upstream and downstream roles in the response to DNA damage. Future research should focus on the mechanism(s) of WRN in the regulation of the various DNA metabolism pathways and development of therapeutic approaches to treat premature aging syndromes such as WS.     

DmWRNexo is a 3′–5′ exonuclease: phenotypic and biochemical characterization of mutants of the Drosophila orthologue of human WRN exonuclease

The premature human ageing Werner’s syndrome is caused by loss or mutation of the WRN helicase/exonuclease. We have recently identified the orthologue of the WRN exonuclease in flies, DmWRNexo, encoded by the CG7670 locus, and showed very high levels of mitotic recombination in a hypomorphic PiggyBac insertional mutant. Here, we report a novel allele of CG7670, with a point mutation resulting in the change of the conserved aspartate (229) to valine. Flies bearing this mutation show levels of mitotic recombination 20-fold higher than wild type. Molecular modelling suggests that D229 lies towards the outside of the molecule distant from the nuclease active site. We have produced recombinant protein of the D229V mutant, assayed its nuclease activity in vitro, and compared activity with that of wild type DmWRNexo and a D162A E164A double active site mutant we have created. We show for the first time that DmWRNexo has 3′–5′ exonuclease activity and that mutation within the presumptive active site disrupts exonuclease activity. Furthermore, we show that the D229V mutant has very limited exonuclease activity in vitro. Using Drosophila, we can therefore analyse WRN exonuclease from enzyme activity in vitro through to fly phenotype, and show that loss of exonuclease activity contributes to genome instability.     

日本血吸虫天冬酰胺酰基内肽酶编码基因表达及诊断应用

目的克隆和表达日本血吸虫天冬酰胺酰基内肽酶（Sj32）,探索该重组抗原在动物血吸虫病诊断方面的应用潜能。方法用PCR法从Sj32前体编码基因中克隆编码成熟体的基因片段,以pET-28（a）为表达载体,用大肠埃希菌表达,制备重组抗原（rSj32）;应用ELISA方法检测待检血清,并比较rSj32、日本血吸虫可溶性虫卵抗原（SEA）以及重组诊断抗原rSj23对人工感染兔、小鼠及水牛血吸虫病的检测效果。结果成功表达了Sj32成熟体,表达产物rSj32的分子量为41 kDa,可用H is柱纯化,得率约为68.8 mg/L培养物。用rSj32检测人工感染兔、小鼠和水牛血清以及对照兔、小鼠和水牛血清,特异性分别为100.0%、96.7%和96.9%,敏感性分别为88.9%、85.0%和71.8%,该抗原对牛血清的敏感性略低于SEA和rSj23,其余检测结果三者相比无显著差异。结论rSj32在诊断动物血吸虫病方面具有研究和应用价值。     

Identification of Taiwan and China-like recombinant avian infectious bronchitis viruses in Taiwan

Infectious bronchitis virus (IBV) infections in poultry cause great economic losses to the poultry industry worldwide. The emergence of viral variants complicates disease control. The IBV strains in Taiwan were clustered into two groups, Taiwan group I and Taiwan group II, based on the S1 gene. A variant was previously identified and showed a distinct S1 gene homology with other local strains. This study investigated the 3′ 7.3 kb genome of eight Taiwan strains isolated from 1992 to 2007. The genes of interest were directly sequenced. Sequence analyses were performed to detect any recombination event among IBVs. The results demonstrated that all of the examined viruses maintained the typical IBV genome organization as 5′-S-3a-3b-E-M-5a-5b-N-UTR-3′. In the phylogenetic analyses, various genes from one strain were clustered into separate groups. Moreover, frequent recombination events were identified in the Simplot analyses among the Taiwan and China CK/CH/LDL/97I-type strains. Putative crossover sites were located in the S1, S2, 3b, M genes and the intergenic region between the M and 5a genes. All of the recombinants showed chimeric IBV genome arrangements originated from Taiwan and China-like parental strains. Field IBVs in Taiwan undergo genetic recombination and evolution.     

基因重组人铁蛋白轻链质控品的制备与评价

目的 构建人铁蛋白轻链基因的原核表达载体,并评价与探讨能在大肠埃希菌(E.coli)中过表达的人铁蛋白轻链所制备的基因rFL质控品的均匀性、稳定性和精密度以及应用价值.方法 采用RT-PCR从人新鲜全血总RNA克隆铁蛋白轻链(ferritin L)基因,构建亚克隆质粒pGM-T/ferritin L;用测序鉴定后,将重组质粒pET-30a/ferritin L转化E.coli BL21(DE3),经异丙基硫代-β-D半乳糖苷(IPTG)诱导rFL表达;采用SDS-PAGE对rFL进行鉴定,用WB分析其抗原性,ACCESS免疫分析仪测定其浓度.同时,采用随机数表从制备的rFL质控品中抽取15支,用单因素方差分析评价质控品的均匀性;用一元线性回归分析评价质控品的稳定性,再根据评价结果计算均匀性和稳定性的不确定度,然后进行精密度评价.结果 构建的铁蛋白轻链原核表达体系经测序显示,扩增的ferritin L基因片段与GenBank中的ferritin L编码序列(NM_000146.3)一致;PCR扩增出与ferritin L DNA的527 bp相符的片段;用IPTG诱导表达后,可得到表达量高的相对分子质量为19 000的rFL,WB证明其具有良好的抗原性;用ACCESS测定rFL稀释1 000倍后的浓度为(1 115.84±38.38) ng/ml.均匀性评价结果显示,高低2个浓度基因rFL质控品样本内和样本间的差异均无统计学意义(高浓度和低浓度基因rFL质控品的F值分别为2.336、0.730,P均>0.05).稳定性结果表明,质控品样本随贮存时间(5个半月)变化的线性趋势差异无统计学意义(F=1.755,P>0.05).精密度评价为高浓度基因rFL质控品批内CV为2.06%,批间CV为2.12%,日间CV为0%,总CV为2.96%;低浓度基因rFL质控品批内CV为2.03%,批间CV为2.08%,日间CV为0%,总CV为2.90%.结论 基因rFL质控品的均匀性、稳定性及精密度符合临床检测要求,可成为具有我国自主知识产权的铁蛋白质控品,同时也为铁蛋白混合质控品的制备提供了原料.

Abstract：

Objective To construct a prokaryotic expression system of human ferritin L which could be overexpressed in E.coli, and then prepare quality control materials of rFL and evaluate the homogeneity, stability, precision and application value of the rFL.Methods RT-PCR was used to clone ferritin L gene with total RNA from peripheral blood.Ferritin L gene was inserted into plasmid pGM-T to construct subclone plasmid pGM-T/ferritin L, which was identified by sequencing.The recombinant plasmid pET-30a/ferritin L was transformed into E.coli BL21 (DE3) and efficiently expressed under IPTG induction.rFL was identified by SDS-PAGE, and its antigenicity was examined by WB.The concentration of rFL was measured by ACCESS immunoassay system.Fifteen control samples were randomly selected using random number table.The homogeneity and stability of rFL were evaluated by one-way analysis of variance (ANOVA) and a linear regression model, respectively.The uncertainties of homogeneity, stability and the precision were evaluated.Results A prokaryotic expression system of ferritin L was successfully constructed.Sequence analysis showed that the ferritin L gene inserted was identical with the one from GenBank(NM_000146.3).The PCR products were 527 bp long, in complete agreement with length of ferritin L gene.The molecular weight of rFL highly expressed in E.coli induced by IPTG was 19 000 Da.The WB analysis indicated that this rFL protein had good antigenicity.The concentration of rFL(1 000 times dilution) measured by ACCESS immunoassay system was (1115.84±38.38) ng/ml.Homogeneity evaluation of low-concentration QC sample and high-concentration QC sample of rFL showed that there were no statistical significances in the within-groups and between-groups (for high-concentration F=2.336, P>0.05 and for low-concentration F=0.730, P>0.05).A linear regression based on the stability test indicated that there was no statistically significant trend of instability in five and a half months (F=1.755, P>0.05). Precision analysis showed the within-run CV, the between-run CV, the between-day CV and the total CV of high-concentration QC sample were 2.06%, 2.12%, 0% and 2.96% respectively; the within-run CV, the between-run CV, the between-day CV and the total CV of low-concentration QC sample 2.03%, 2.08%, 0% and 2.90%, respectively.Conclusion This rFL for quality control materials with independent intellectual property rights could meet the clinical requirement of homogeneity, stability and precision, and could provide raw materials for preparation of mixed ferritin for quality control.     

综合医院与妇产科医院合并重组对住院工作量的影响

目的探讨医院合并重组对住院工作量的影响。方法计算历年出院人数及实际占用床日定基比,绘制工作量变动趋势图,对合并后的成都市第九人民医院、妇产科医院住院工作量变动趋势进行分析。结果(1)两院合并后的2001年～2003年,处于磨合期,住院工作量增幅相对平缓;2004年～2007年进入快速上升期,住院工作量指标增幅达合并初期的1.1倍。(2)作为该院重点学科的产科,其住院工作量一直处于各科之首,2004年～2007年,上升趋势明显。结论成都市坚持卫生资源优势互补的原则对医疗机构进行合并重组,收到了优化配置医疗卫生资源,提高医院运行效率的预期效果。     

Diversity and evolution of MHII β genes in a non-model percid species—The Eurasian perch (Perca fluviatilis L.)

This study provides the first investigation of the diversity, structure, and molecular evolution of MHII β genes in a non-model percid species – the Eurasian perch (Perca fluviatilis L.). PCR primers developed here were highly specific, and documented a high diversity of the MHII β1 domain in perch. Our results suggest a minimum of eight MHII β loci in this species – a finding congruent with several studies suggesting that many Euteleostei posses multiple MHII β loci. As for other vertebrates, both positive selection and gene-conversion contribute to the reported high allelic diversity. Similarly, the MHII β1 domain in perch exhibits a characteristic MHC fold known from other vertebrates. In addition, our results suggest some teleost specific differences of the MHII β1 domain, including: differences in chemical properties of specific amino acids in the β1 domain, the absence of the tetrapod specific glycolisation signal, and differences in the positions of some of the positively selected codons in the MHII β1 domain, which are presumably involved in antigen binding. Future studies should investigate the teleost MHII β genes in more details in order to confirm the suggested differences, and to determine the extent to which these differences prevail in different teleost lineages.     

可溶性AZURIN分子重组子构建对人骨肉瘤U2OS细胞的杀伤和诱导凋亡

目的:研究可溶性细菌氧化还原蛋白AZURIN对人骨肉瘤U2OS细胞的生长抑制效应及凋亡诱导作用.方法:用PCR方法由假单胞菌染色体DNA扩增AZURIN基因片段,将其融合插入原核表达载体pQE30中,并在M15大肠杆菌表达可溶性的AZURIN蛋白.该蛋白经纯化和复性后,诱导人骨肉瘤U2OS细胞,通过MTT法、Hoechest 33258荧光染色法及倒置显微镜观察凋亡细胞;流式细胞仪分析细胞周期、凋亡率、线粒体膜电位变化和DNA断裂实验检测细胞增殖和细胞凋亡.结果:AZURIN的蛋白纯度达99.1%以上.用50～200 mg/L的蛋白诱导人骨肉瘤U2OS细胞12 h以上细胞的生长和增殖被显著抑制,并且观察到典型的凋亡细胞、凋亡小体、凋亡峰及DNA的片段化等凋亡的特征性变化,线粒体跨膜电位随着AZURIN作用的时间和浓度的增加而逐渐降低,对照组和50 mg/L AZURIN、100 mg/L AZURIN、200 mg/L AZURIN组细胞线粒体跨膜电位分别降低(7.76±0.47)%、(9.01±0.87)%、(24.13±2.12)%、(37.48±3.59)%(P<0.01).细胞的凋亡率也呈剂量依赖和时间依赖关系,作用48 h时凋亡率达峰值(35.8±3.78)%.结论:利用大肠杆菌制备的小分子蛋白AZURIN对人骨肉瘤U2OS细胞有明显的增殖抑制效应和凋亡诱导作用,线粒体途径可能是AZURIN诱导细胞凋亡的机制之一.     

Energy-dependent mitochondrial mutagenicity of antibacterial ofloxacin and its recombinogenic activity in yeast

Ofloxacin, a specific inhibitor of bacterial topoisomerase II, is known to inhibit the growth of yeast cells and to induce rho ^– mutants in the yeast S. cerevisiae. The frequency of ofloxacin-induced petite mutants under non-growth conditions was found to be strongly diminished when the cells were depleted in intramitochondrial ATP. Under optimal conditions of mitochondrial mutagenesis the drug induced mitotic recombination and reverse mutation in diploid strains but failed to cure either killer plasmids or the 2 m DNA of dividing cells. The sensitivity to ofloxacin of the strains deficient in the DNA strandbreak repair pathway (rad52) was significantly higher then that of the wild-type strains and of the mutants deficient in excision or mutagenic DNA repair. The results are compatible with the idea that the cytotoxic and genetic activity of ofloxacin in yeast probably results from the inhibited DNA ligation function of topoisomerase II creating DNA breaks that are reparable through the recombination repair pathway.     

Unusual organization and lack of recombination in the ribosomal RNA genes of Coprinus cinereus

Summary We find three interesting characteristics of the genes encoding the ribosomal RNA (rRNA) in the basidiomycete Coprinus cinereus. First, there are only 60 to 90 copies of the genes, fewer than in other fungi. Second, the genes are organized in an unusual arrangement. The 5S rRNA genes are located in the repeat unit which encodes the other rRNAs and all four rRNAs are transcribed in the same direction. Third, meiotic recombination is inhibited within the ribosomal DNA.