Anonymous nuclear DNA markers in the American oyster and their implications for the heterozygote deficiency phenomenon in marine bivalves

A puzzling population-genetic phenomenon widely reported in allozyme surveys of marine bivalves is the occurrence of heterozygote deficits relative to Hardy-Weinberg expectations. Possible explanations for this pattern are categorized with respect to whether the effects should be confined to protein-level assays or are genomically pervasive and expected to be registered in both protein- and DNA-level assays. Anonymous nuclear DNA markers from the American oyster were employed to reexamine the phenomenon. In assays based on the polymerase chain reaction (PCR), two DNA-level processes were encountered that can lead to artifactual genotypic scorings: (a) differential amplification of alleles at a target locus and (b) amplification from multiple paralogous loci. We describe symptoms of these complications and prescribe methods that should generally help to ameliorate them. When artifactual scorings at two anonymous DNA loci in the American oyster were corrected, Hardy-Weinberg deviations registered in preliminary population assays decreased to nonsignificant values. Implications of these findings for the heterozygote-deficit phenomenon in marine bivalves, and for the general development and use of PCR-based assays, are discussed.      

Basolateral plasma membrane localization of ouabain-sensitive sodium transport sites in the secretory epithelium of the avian salt gland

The distribution of Na+ pump sites (Na+-K+-ATPase) in the secretory epithelium of the avian salt gland was demonstrated by freeze-dry autoradiographic analysis of [(3)H] ouabain binding sites. Kinetic studies indicated that near saturation of tissue binding sites occurred when slices of salt glands from salt-stressed ducks were exposed to 2.2 μM ouabain (containing 5 μCi/ml [(3)H]ouabain) for 90 min. Washing with label-free Ringer's solution for 90 min extracted only 10% of the inhibitor, an amount which corresponded to ouabain present in the tissue spaces labeled by [(14)C]insulin. Increasing the KCl concentration of the incubation medium reduced the rate of ouabain binding but not the maximal amount bound. In contrast to the low level of ouabain binding to salt glands of ducks maintained on a freshwater regimen, exposure to a salt water diet led to a more than threefold increase in binding within 9-11 days. This increase paralleled the similar increment in Na+-K+-ATPase activity described previously. [(3)H]ouabain binding sites were localized autoradiographically to the folded basolateral plasma membrane of the principal secretory cells. The luminal surfaces of these cells were unlabeled. Mitotically active peripheral cells were also unlabeled. The cell-specific pattern of [(3)H]ouabain binding to principal secretory cells and the membrane-specific localization of binding sites to the nonluminal surfaces of these cells were identical to the distribution of Na+-K+-ATPase as reflected by the cytochemical localization of ouabain-sensitive and K+-dependent nitrophenyl phosphatase activity. The relationship between the nonluminal localization of Na+-K+-ATPase and the possible role of the enzyme n NaCl secretion is considered in the light of physiological data on electrolyte transport in salt glands and other secretory epithelia.     

The 3'' untranslated regions of two related mRNAs contain an element highly repeated in the sea urchin genome.

Two closely related cDNA clones, pSpec1 and pSpec2, specifying two developmentally regulated tissue specific mRNAs from sea urchin embryos were used to probe a sea urchin genomic lambda library. Screening 10,000 phage by plaque hybridization yielded several hundred positive signals. With more stringent wash procedures, only two to three phage were positive. Three of these phage, one isolated by stringent wash procedures and two isolated by standard wash procedures were further investigated by restriction analysis, RNA gel blots, and DNA sequencing. The phage isolated by the stringent wash procedure appears to be a gene coding for the Specl mRNA. The other phage contain only partial homology to pSpec1 and pSpec2, 150 to 200 base pairs of the 3' untranslated region of the Spec1 and Spec2 mRNAs. It is concluded that the Spec1 and Spec2 mRNAs contain a highly repetitive element near their 3' end. The element is present at 2000 to 3000 copies per genome and may be transcribed at some sites other than those coding for the Spec1 and Spec2 genes. The possible function and evolutionary origin of the repetitive element is discussed.     

构建基于CRISPR/Cas9技术敲除ALOX5的质粒

旨在利用CRISPR/Cas9技术构建敲除花生四烯5-脂氧合酶基因(Arachidonate 5-lipoxygenase gene,ALOX5)的重组质粒。设计合成3对靶向敲除ALOX5第六外显子的sgRNA,将其分别插入到CRISPR/Cas9质粒骨架pX458载体中,转化感受态大肠杆菌DH5α后挑取克隆,通过测序评估重组质粒是否构建成功。将构建好的重组质粒转染293T细胞,在荧光显微镜下观察转染效果,挑取转染成功的细胞,用试剂盒提取转染细胞基因组DNA,PCR扩增含敲除位点的DNA片段,用测序技术获得核苷酸序列,用DNAStar软件分析转染细胞中ALOX5基因敲除情况。测序结果表明2对双链sgRNA寡核苷酸已插入质粒,且序列正确,靶向ALOX5基因的重组质粒pX458-sgRNAs-ALOX5构建成功。其在293T细胞中的转染效率约为50%,用一代测序法未检测到sgRNAs的切割效果。初步表明利用CRISPR/Cas9技术成功构建靶向ALOX5基因的重组质粒pX458-sgRNAs-ALOX5。     

羌活与宽叶羌活的群体遗传结构和种间分化研究

为了合理利用羌活和宽叶羌活的药用植物资源,同时保护其物种多样性,该研究利用SSR分子标记技术对羌活与宽叶羌活邻域及异域分布的23个自然种群,共计227个个体进行多样性和种间分化研究。结果显示:(1)两个物种具有中等水平的遗传多样性;羌活的平均等位基因数(N_a)、有效等位基因数(N_e)和期望杂合度(H_e)分别为2.603、1.777和0.313,均高于宽叶羌活(分别为2.200、1.641和0.308)。(2)分子方差分析表明,两个物种的遗传变异主要存在于群体内,羌活和宽叶羌活群体间分化系数分别为0.181和0.191。(3)Structure聚类分析和主坐标分析(PCoA)将所有取样个体分为两大遗传组分,分别对应于羌活和宽叶羌活两个物种,二者间存在着有限的基因交流。研究表明,羌活与宽叶羌活物种间存在较高程度的遗传分化,并且遗传变异主要源自群体内,应各自划分为不同的地理单元进行多样性保护。     

中国三种常见蒿属植物潜在地理分布及其主导气候因子

裂叶蒿(Artemisia tanacetifolia)、大籽蒿(Artemisia sieversiana)和艾(Artemisia argyi)是我国常见的蒿属(Artemisia)植物,其分布区域遍布全国。本文利用MaxEnt模型预测3种蒿属植物在当前气候条件以及未来两种气候情景下的潜在分布区。采用受试者工作特征曲线(receiver operating characteristic curve,ROC)检验模型精度。训练数据和测试数据的AUC值均在0.8以上,表明预测结果可靠性良好。在当前的气候条件下,裂叶蒿最适分布区主要为黄土高原、内蒙古高原和东北平原;大籽蒿的最适分布区为西藏南部谷地、横断山地区、黄土高原、内蒙古高原和东北平原;艾的最适分布区有两个,一个位于台湾岛南部,另一个为大巴山、巫山、云贵高原北部、黄土高原和东北平原南部区域。2070年RCP2.6和RCP8.5情景下,裂叶蒿及大籽蒿的高适宜区面积减小,艾的最适分布区面积增加。Jackknife检验结果表明,年均降水量是预测裂叶蒿分布最有效的气候因子,5月降水是预测大籽蒿分布的最显著的气候因子,8月水汽压对艾的影响最大。本研究结果为蒿属植物资源的合理利用提供了科学依据。     

The early life history of two sympatric New Zealand octopuses: eggs and paralarvae of Octopus huttoni and Pinnoctopus cordiformis

This study combined morphological and morphometric information on egg clutches, egg capsules and paralarvae of two sympatric coastal octopuses from New Zealand waters, Octopus huttoni and Pinnoctopus cordiformis, to provide species-specific traits to identify their early life stages obtained from field surveys. Eggs of O. huttoni (2.5 mm length; 1 mm width) were entwined with one another forming strings that ranged from 11 to 25.8 mm in length. Eggs of P. cordiformis (6.4 mm length; 1.5 mm width) were significantly bigger than those of O. huttoni and were grouped in small clusters of about seven eggs. Paralarvae O. huttoni and P. cordiformis differed in hatching size (1.4 mm versus 3.1 mm mantle length), number of suckers per arm (four versus eight), number of lamellae per outer demibranch (five versus ten) and arrangements of chromatophores in the body surface (29 to 59 versus 91 to 179), respectively. The morphological traits described in hatchlings from the laboratory allowed comparisons with field-collected paralarvae, suggesting that such characters were reliable species-specific patterns to enable a consistent differentiation between the early life stages of these two sympatric species, even in the absence of the brooding female.     

长爪沙鼠肝细胞体外分离培养体系的建立

目的建立长爪沙鼠原代肝细胞分离培养体系。方法以雄性长爪沙鼠为供体,采用组织消化法和Seglen两步灌流法分离肝细胞,以台盼蓝染色检测细胞得率和活率,过碘酸-希夫氏反应(PAS)鉴定肝细胞,倒置显微镜观察肝细胞形态变化,并使用含有多种细胞因子的培养基维持培养。结果组织消化法和Seglen两步灌流法平均每只长爪沙鼠可分别获得肝细胞(1.33±0.34)×107个、(3.97±1.15)×107个,细胞活率分别为(29.4±6.05)%、(80.3±4.56)%,这两种方法在细胞得率及活率方面存在显著差异。肝细胞内因有大量的糖原颗粒,经PAS染色后被染成红色。结果表明肝细胞在贴壁后72 h内,肝细胞形态发生显著变化。结论采用胶原酶经肝门静脉灌流分离肝细胞是一种高效获得肝细胞的方法。各种细胞因子有利于维持肝细胞在体外的生长分化,长爪沙鼠原代肝细胞分离培养体系的建立将为肝脏相关疾病研究和防治药物的开发提供技术支持。