无线粒体DNA的人肺腺癌A549细胞系的构建

目的:建立无线粒体DNA(mtDNA)的人肺腺癌ρ~0A549细胞系。方法:在含50 ng/mL溴化乙锭(EB)、100μg/mL丙酮酸钠和50μg/mL尿嘧啶核苷的RPMI1640细胞培养基中传代培养A549细胞;用低剂量EB连续诱导培养35 d后,采用光镜观察、TaqMan探针法实时荧光定量PCR(qPCR)和Western印迹鉴定无mtDNA的ρ~0A549细胞系;采用MTT法测定ρ~0A549细胞增殖曲线。结果:倒置显微镜下野生型ρ^+A549细胞为多角形,ρ~0A549细胞形态呈拉长枝状;qPCR结果显示,低剂量EB诱导35 d的ρ~0A549细胞中无mtDNA的存在。Western印迹结果显示,ρ^+A549细胞中能表达核基因编码的线粒体蛋白SDHA和ATP5A,也能表达线粒体基因组编码的蛋白MT-COXI和MT-ATP6;ρ~0A549细胞中无MT-COXI和MT-ATP6蛋白表达,但核基因编码的SDHA和ATP5A蛋白能够正常表达。MTT结果显示,与ρ^+A549细胞相比,ρ~0A549细胞生长速度明显减慢,差异有统计学意义(P<0.05)。结论:构建和鉴定了无mtDNA的人肺腺癌ρ~0A549细胞系,为后续探讨mtDNA缺失或突变与人肺腺癌发生之间的关系奠定了实验基础。     

Real-time detection of Escherichia coli O157:H7 sequences using a circulating-flow system of quartz crystal microbalance

A DNA piezoelectric biosensing method for real-time detection of Escherichia coli O157:H7 in a circulating-flow system was developed in this study. Specific probes [a 30-mer oligonucleotide with or without additional 12 deoxythymidine 5′-monophosphate (12-dT)] for the detection of E. coli O157:H7 gene eaeA, synthetic oligonucleotide targets (30 and 104 mer) and PCR-amplified DNA fragments from the E. coli O157:H7 eaeA gene (104 bp), were used to evaluate the efficiency of the probe immobilization and hybridization with target DNA in the circulating-flow quartz crystal microbalance (QCM) device. It was found that thiol modification on the 5′-end of the probes was essential for probe immobilization on the gold surface of the QCM device. The addition of 12-dT to the probes as a spacer, significantly enhanced (P < 0.05) the hybridization efficiency (H%). The results indicate that the spacer enhanced the H% by 1.4- and 2-fold when the probes were hybridized with 30- and 104-mer targets, respectively. The spacer reduced steric interference of the support on the hybridization behavior of immobilized oligonucleotides, especially when the probes hybridized with relatively long oligonucleotide targets. The QCM system was also applied in the detection of PCR-amplified DNA from real samples of E. coli O157:H7. The resultant H% of the PCR-amplified double-strand DNA was comparable to that of the synthetic target T-104AS, a single-strand DNA. The piezoelectric biosensing system has potential for further applications. This approach lays the groundwork for incorporating the method into an integrated system for rapid PCR-based DNA analysis.     

HBV包膜-核心蛋白融合基因DNA疫苗诱导小鼠持久的细胞免疫应答

构建编码HBV包膜-核心蛋白融合基因的DNA疫苗pSC、pSS1S2C和编码HBV包膜蛋白或核心蛋白基因的DNA疫苗pHBs、pHBc,分别肌肉注射免疫BALB/c小鼠,检测小鼠的血清抗体、T细胞增殖和细胞毒性T淋巴细胞反应,比较融合基因DNA疫苗与单基因DNA疫苗诱生免疫应答的强度,发现融合基因DNA疫苗诱生抗体的效率明显不及单基因DNA疫苗,但其能诱导更强、更持久的细胞免疫应答,表明HBV包膜-核心蛋白融合基因DNA疫苗对于治疗慢性乙型肝炎可能比单基因DNA疫苗更为有效.     

花生种子高纯度DNA的提取（简报）

花生种子高纯度ＤＮＡ的提取（简报）王艳,何军贤,傅家瑞（中山大学生命科学学院,广州５０２７５）关键词花生;种子;ＤＮＡ;十六烷基三甲基溴化铵ＲＡＰＩＤＡＮＤＥＦＦＩＣＩＥＮＴＰＵＲＩＦＩＣＡＴＩＯＮＯＦＤＮＡＦＲＯＭＰＥＡＮＵＴ（ＡＲＡＣＨＩＳＨＹＰ...     

Kraftionema allantoideum,a new genus and family of Ulotrichales (Chlorophyta) adapted for survival in high intertidal pools

The marine, sand‐dwelling green alga Kraftionema allantoideum gen. et sp. nov. is described from clonal cultures established from samples collected in coastal, high intertidal pools from south eastern Australia. The species forms microscopic, uniseriate, unbranched, 6–8 μm wide filaments surrounded by a gelatinous capsule of varying thickness. Filaments are twisted, knotted, and variable in length from 4 to 50 cells in field samples but straighter and much longer in culture, up to 1.5 mm in length. Cell division occurs in several planes, resulting in daughter cells of varying shape, from square to rectangular to triangular, giving rise to gnarled filaments. Mature cells become allantoid, elongate with rounded ends, before dividing one time to form bicells comprised of two domed cells. Adjacent bicells separate from one another and mature filaments appeared as a string of loosely arranged sausages. A massive, single, banded chloroplast covered 3/4 of the wall circumference, and contained a single large pyrenoid encased in a starch envelope that measures 1.5–2.5 μm. Filaments were not adhesive nor did they produce specialized adhesive cells or structures. Reproduction was by fragmentation with all cells capable of producing a new filament. No motile or reproductive cells were observed. Filaments in culture grew equally well in freshwater or marine media, as well as at high salinity, and cells quickly recovered from desiccation. Phylogenetic analysis based on the nuclear‐encoded small subunit ribosomal RNA (18S) shows the early branching nature of the Kraftionema lineage among Ulotrichales, warranting its recognition as a family (Kraftionemaceae).     

从12S rRNA基因片段序列研究20种蛇的系统发生关系

本文测定了中国产蛇亚目20种蛇约800bp的mtDNA 12S rDNA基因片段序列。所测序列与楔齿蜥的同源序列一起经Clustal X1.8软件比对,共有881个位点,其中变异位点有494个。以楔齿蜥为外群,用NJ法构建了4科20种蛇的进化关系树,对这20种蛇的系统发生关系作了初步探讨。研究结果表明：所研究的4个科20种蛇分成4个支系。第一个支系包括蟒科的东方沙蟒和蟒2种蛇;第二个支系为蝰科3种蛇,即草原蝰、尖吻蝮、竹叶青组成一个单系群;眼镜蛇科的眼镜蛇和银环蛇构成第三个支系;游蛇科的13种蛇构成了第四个支系,其中灰鼠蛇、乌梢蛇和赤链蛇组成一个支系,锦蛇属的7种蛇组成一个单系群,然后它们与前一支系相聚。剩下的颈槽蛇属两种聚类后与赤链华游蛇构成一个支系,并与游蛇科其它蛇组成姐妹群。第四支系首先与第三支系眼镜蛇科聚类,第二支系蝰科构成了三、四支系眼镜蛇科和游蛇科的姐妹群,第一支系蟒科在系统树的最基部,为四个科中较原始的类群。     

Genetic monogamy in blue-headed vireos and a comparison with a sympatric vireo with extrapair paternity

Based on the breeding synchrony hypothesis, we predicted, intwo congeners that nest in simiilar habitat but differ in nestingsynchrony, that blue-headed vireos (Vireo solitarius) wouldhave fewer extrapair fertilizations (EPFs) thaii red-eyed vireos(V. olivaceus EPFs were rare in blue-headed vireos (1/37 nestlings),but common in red-eyed vireos (11/19 nestlings). We studiedthe behavior of blue-headed vireos to determine what factorscould promote genetic monogamy. We found no evidence that malesmate guarded to prevent extrapair copulations from occurring.Males did not follow fertile mates closely when mates left thenest (14–25% of female departures) and, during the egg-layingperiod, males were often alone on the nest (22.3 mm/h). Femaleblue-headed vireos, but not red-eyed vireos, obtain direct benefitsfrom social mates such as nest building and incubation (49.1%of the total), and they assess male quality long before becomingfertile. Female blue-headed vireos spent more time incubatingwhen their mates had low incubation effort. Furthermore, maleincubation effort was positively correlated with nest survivalduring incubation. We discuss the evolution of genetic monogamyand sex role convergence in blue-headed vireos in relation toasynchronous breeding.     

Hot topics in epigenetic mechanisms of aging: 2011

Aging is a complex process that results in compromised biological functions of the organism and increased susceptibility to disease and death. Although the molecular basis of aging is currently being investigated in many experimental contexts, there is no consensus theory to fully explain the aging process. Epigenetic factors, including DNA methylation, histone modifications, and microRNA expression, may play central roles in controlling changes in gene expression and genomic instability during aging. In this Hot Topic review, we first examine the mechanisms by which these epigenetic factors contribute to aging in diverse eukaryotic species including experimental models of yeasts, worms, and mammals. In a second section, we will emphasize in the mammalian epigenetic alterations and how they may affect human longevity by altering stem cell function and/or somatic cell decline. The field of aging epigenetics is ripe with potential, but is still in its infancy, as new layers of complexity are emerging in the epigenetic network. As an example, we are only beginning to understand the relevance of non-coding genome to organism aging or the existence of an epigenetic memory with transgenerational inheritance. Addressing these topics will be fundamental for exploiting epigenetics phenomena as markers of aging-related diseases or as therapeutic targets.     

Genetic and alkaloid analysis of Menispermum dauricum DC. by RAPD and HPLC

The aim of the present work was to determine whether dauricine could be used as a taxonomic marker for Menispermum dauricum DC., and to explore the correlation among RAPD, ecological markers and chemical markers. To this end, the chemical and genetic differences of 173 individual samples of M. dauricum from nine different sources were studied based on the relevant ecological factors including longitude, latitude, annual precipitation, mean temperature, annual accumulated temperature and mean sea level. The contents of dauricine in the sample rhizomes were assayed by HPLC with photodiode array detection. The leaves from the same sample were assayed using randomly amplified polymorphism DNA (RAPD). The genetic distances were then compared. Hierarchical cluster analysis and multiple linear stepwise regression analysis were used in the statistical analysis. The results indicated that the contents of dauricine were respectively correlated with the genetic distance (r = 1.000), longitude (r = 0.849), latitude (r = 0.861), annual precipitation (r = 0.903), mean temperature(r = 0.912), annual accumulated temperature (r = 0.919) and mean sea level (r = 0.925). It is concluded that the content of dauricine in M. dauricum is significantly correlated with genetic distance and ecological factors, and may be used as the taxonomic marker.     

Cytosolic RNA:DNA hybrids activate the cGAS–STING axis

Intracellular recognition of non‐self and also self‐nucleic acids can result in the initiation of potent pro‐inflammatory and antiviral cytokine responses. Most recently, cGAS was shown to be critical for the recognition of cytoplasmic dsDNA. Binding of dsDNA to cGAS results in the synthesis of cGAMP(2′–5′), which then binds to the endoplasmic reticulum resident protein STING. This initiates a signaling cascade that triggers the induction of an antiviral immune response. While most studies on intracellular nucleic acids have focused on dsRNA or dsDNA, it has remained unexplored whether cytosolic RNA:DNA hybrids are also sensed by the innate immune system. Studying synthetic RNA:DNA hybrids, we indeed observed a strong type I interferon response upon cytosolic delivery of this class of molecule. Studies in THP‐1 knockout cells revealed that the recognition of RNA:DNA hybrids is completely attributable to the cGAS–STING pathway. Moreover, in vitro studies showed that recombinant cGAS produced cGAMP upon RNA:DNA hybrid recognition. Altogether, our results introduce RNA:DNA hybrids as a novel class of intracellular PAMP molecules and describe an alternative cGAS ligand next to dsDNA.