噬热栖热菌质粒分离蛋白同系物ParBTth的表达与重组酶活性

在低拷贝质粒中,质粒分离基因位点parABS对质粒DNA的子细胞精确分离有重要意义。噬热栖热菌(Thermus thermophilus)为多倍体,其染色体上表达有一个parABS同系物,命名为parABSTth,包含parATth和parBTth两个基因,以及一个候选parSTth的DNA序列(5’-TGTTTCCCGTGAAACA-3’)。parABSTth基因位点的功能与机制尚未清楚。本研究首先在大肠杆菌Escherichia coli中克隆并表达了T. thermophilus parABSTth位点中的parBTth基因。利用Ni-IDA亲和层析方法纯化得到了重组蛋白ParBTth;经SDS-PAGE测定,该重组蛋白纯度达到了近99%,通过凝胶迁移实验(EMSA)验证了重组ParBTth蛋白的功能。结果显示,重组ParBTth能与候选parSTthDNA序列特异性相结合。因此,异源表达的重组ParBTth蛋白是具有生物学活性的。该结果也验证了候选parSTthDNA序列确实为T. thermophilus的parABSTth基因位点中特异性的parS序列。本研究为揭示多倍体细菌染色体的子细胞分离过程原理提供了一定的基础理论依据。     

影响pBR322质粒DNA对E. Col I C600转化的因子

在DNA体外重组技术的研究和应用中，转 化是个很重要的步骤。自Cohen等人^[2]在1972 年首次用CaCl：处理的方法成功地进行了R因 子对E. colt C600的转化之后，这个方法一直 广泛地应用于大肠杆菌各受体菌系的转化。鼠 伤寒沙门氏杆菌S. typhimurium^[3]和金黄色葡 萄球菌S. aureus^[4]，的转化也都沿用此法。直 至1978年，Michael等^[5]在用pBR 322质粒 DNA对E. colt X1776的转化中却采用了与 此不同的方法。     

快速鉴定和大量纯化质粒DNA的方法

为了快速鉴定重组质粒DNA, Birnboim等 人^[1]建立了快速碱性抽提法。在此基础上,我 们作了某些修改,不仅较有效地防止了质粒 DNA变性形式的出现,而且可以用来大量抽提 质粒DNA。若再协同轻基磷灰石柱层析121和酸 酚法^[6]处理,可获得纯度较高的质粒DNA。我 们用修改法提取了分子量从2.6X10^[6]-26X10^[6] 道尔顿的4种不同质粒DNA,均收到了比较理 想的结果,特别对于难于抽提的或大质粒DNA 更为有效。     

琼脂糖凝胶电泳分离纯化和制备质粒DNA

分离纯化质粒DNA,在遗传工程和质粒的 分子遗传学研究中是重要的一环。分离和制备 质粒DNA的方法很多,有：澳化乙锭密度梯度 祛^[2-5]、两相法^[6]、酸酚法^[7][碱变性法^[8]p、甲基化 白蛋白（MAK）柱层析法^[9]硝酸纤维素法^[10]、电 泳法^[11]等,这些方法都各有所长。根据质粒的超 卷曲结构、开环结构和线状结构的不同,各个质 粒分子量大小的不同,以及质粒DNA与RNA 和染色体DNA在电泳中迁移率的不同,在我 们实验室的条件下,研制了一种琼脂糖凝胶电 泳分离纯化和制备质粒DNA装置。运用这种 装置,纯化制备了以下质粒： pBR322, pASI, pUB110、F}lac+proA+B+, pVA517A, pVA517B, pVA517C, pVA517D, pVA517E, pVA517F, pVA517G和pVA517H。并且对它们进行了电 镜观察,对其中纯化过的pBR322和pASI质粒 进行了转化实验,证明它们有生物活性。同时, 应用这种装置,可以一次分离具有不同分子量 大小的几种质粒,回收率约为85多。结果表明, 这种装置结构简单,操作方便,是纯化制备质粒 DNA的一种可用工具。     

非洲钝缘蜱感染Q热立克次体敏感度的测定

Q热立克次体以10^-1—10^-12不同稀释度,用血腔注射法感染非洲钝缘蜱,感染35天,取蜱血淋巴液,分别用Gimenez染色法及免疫荧光技术检查,在10^-1—10^-5的稀释度,两种方法检出率基本相似。10^-6以下各组,免疫荧光尚可检出少数阳性标本,而Gimcnez法则无阳性,前者总阳性率为48.28%,后者为40.68%。感染的蜱悬液接种豚鼠,12组动物血清做Q热补体结合试验全部阳性。动物发病情况如潜伏期长短、发烧天数等似与蜱内立克次体的含量有关。     

金属离子抑制细菌整合子捕获耐药基因盒的机制研究

【目的】探索金属离子对整合子捕获耐药基因盒的影响及其相关机制。【方法】在大肠埃希菌中构建一个1类整合子捕获耐药基因盒的体内模型。通过实时荧光定量PCR(real-time fluorescence quantitative PCR,RT-qPCR)测定不同浓度银(0.3、0.9、1.5μg/mL的Ag+)和铜离子(5、50、100、150、210μg/mL的Cu²⁺)干预后实验组和无金属离子干预对照组整合子整合频率,并用表型筛选法验证。利用质谱法测量细菌吸收的金属离子浓度,并进一步通过转录组测序方法分析银离子抑制细菌整合子捕获耐药基因盒的分子机制。【结果】qPCR和表型筛选法的结果表明,0.9μg/mL银离子组整合频率为9.42×10^-5 (6.49×10^-5,1.44×10^-4),1.5μg/mL银离子组整合频率为7.29×10^-5 (4.45×10^-5,9.03×10^-5),与对照组2.59×10^-4 (2....     

利用噬菌体展示技术构建Mhp168株Hsp70随机肽库

[目的]构建Mhp168株Hsp70 C端基因的噬菌体展示随机肽库。[方法]扩增猪肺炎支原体168株热休克蛋白Hsp70 C端1 803 bp的DNA序列。回收Hsp70 DNaseⅠ消化产物中80～150 bp的DNA片段与p C89载体连接。辅助噬菌体超感染转化重组噬粒的E.coli XL1-Blue细胞。检测肽库的多样性,测定库容。[结果]在噬菌体表面展示出Hsp70 C端基因的随机片段融合蛋白。随机肽库库容约为9.9×10~3,滴度约为1.5×10~(13)PFU/ml。[结论]所构建的随机肽库可以满足Hsp70构象型抗原表位筛选。     

金属离子促进嗜热四膜虫错配修复蛋白Mlh3核酸内切酶活性

嗜热四膜虫有性生殖过程中生殖系小核延伸并活跃转录,减数分裂过程中染色体同源重组起始于程序化的DNA双链断裂的形成,DNA错配修复系统能够去除DNA复制过程中所引起的错配并促进同源重组。减数分裂特异表达的错配修复因子Mlh3对四膜虫的有性生殖是必需的,然而具体功能并不清楚。本研究人工合成MLH3（TTHERM_001044369）基因,构建重组表达质粒pGEX-MLH3, 转化大肠杆菌BL21（DE3）并获得重组表达的GST-Mlh3蛋白。纯化的GST-Mlh3蛋白在配位不同的金属离子Cu2+、Mn2+、Mg2+后,有效切割超螺旋质粒DNA。ATP和ADP可进一步促进Mlh3的核酸内切酶活性。突变Mlh3中离子结合模体DQHA(X)2E(E)4E中的D117和E123位点,Mlh3D117N/E123A的核酸内切酶活性降低。进一步删除离子结合和ATP结合位点的C端结构域,突变体的核酸内切酶活性进一步降低,表明Mlh3的核酸内切酶活性是离子依赖型。减数分裂期HA-Mlh3免疫共沉淀鉴定了Mlh3可能的相互作用因子链交换蛋白Dmc1、DSB修复蛋白Mnd1、MutS、染色体维持蛋白Smc2和Smc4。结果表明,四膜虫的Mlh3通过金属离子依赖的内切酶活性,以及与其他因子相互作用,在减数分裂错配修复和同源重组过程中发挥重要作用。     

pUB 110质粒转化枯草杆菌Ki-2株及其突变体的研究

迄今文献中报道枯草杆菌基因克隆化都采用枯草杆菌168株及其突变体。本文采用我国分离的枯草杆菌Ki-2株及其突变体Ki-2-1 32(Thr~-Ile~-Val~-)和Ki-2-148(ura~-)为pUB110质粒DNA的受体菌株。用酸酚法提纯pUB110质粒DNA,在琼脂糖凝胶电泳上看不到样品中有染色体DNA的带。结果表明,Ki-2、Ki-2-132和Ki-2-148都可作pUB 110质粒的受体菌,其转化频率在10~(—3)—10~(—8)之间,因菌株和条件不同,频率有所差异。Ki-2-132的转化效率为每微克DNA可产生10~4转化体。pUB 110质粒DNA浓度在0.01—1.00μg/ml之间时,转化体数目随DNA浓度增加而增加,其中,在浓度为0.01—0.1μg/ml之间成直线关系,测定的DNA依赖指数为1.04,系一级反应。从pUB110质粒DNA转化168、Ki-2、Ki-2-132、Ki-2-148的转化体中提取的质粒DNA仍具有pUB110质粒DNA的抗卡那霉素的转化活性。从转化体提纯的质粒DNA的电泳图以及EcoRI消化后的电泳图与原来的pUB110 DNA的电泳图相同。     

嗜热厌氧乙醇菌JW200转化条件的研究

摘要 嗜热厌氧乙醇菌遗传转化系统的缺少,制约了对该菌理论基础和应用领域的进一步研究。利用聚乙二醇（PEG6000）转化和电转化技术国际首次实现了嗜热厌氧乙醇菌JW200外源基因的导入。PEG转化效率很低,因此选择对电转化条件进行优化,转化效率从4±3.2个转化子/μg质粒DNA提高到50±7.4个转化子/μg质粒DNA。实验表明获得较高的转化效率的必要条件是在细胞密度为OD660 0.2时添加甘氨酸与蔗糖后继续培养2h以及细胞在电击前的收集与洗涤保持低温。本研究为利用基因工程手段改造嗜热厌氧乙醇菌和从分子水平研究胞内乙醇代谢途径奠定了基础。     

The simple-septate basidiomycetes: a synopsis

The simple-septate basidiomycetes comprise more than 8,000 species that show a high morphological and ecological heterogeneity. To gain insight in the phylogenetic relationships within this group, we compared several ultrastructural features such as septal pore apparatus, form, and behavior of the spindle pole bodies, types of host–parasite interaction, presence or absence of colacosomes, symplechosomes, atractosomes, and cystosomes as well as nuclear rDNA sequences coding for small- and large-subunit rRNA. Based on our integrated analysis, we propose a new classification system for the simple-septate basidiomycetes with the subphylum Pucciniomycotina and the classes Agaricostilbomycetes, Atractiellomycetes, Classiculomycetes, Cryptomycocolacomycetes, Cystobasidiomycetes, Microbotryomycetes, Mixiomycetes, and Pucciniomycetes. We also propose the pucciniomycotinous taxa Cystobasidiales, Erythrobasidiales, Helicobasidiales, Mixiales, Naohideales, Pachnocybales, Spiculogloeales, and Kondoaceae and the new subphyla Agaricomycotina (equivalent to the current Hymenomycetes) and Ustilaginomycotina (equivalent to the current Ustilaginomycetes).     

Unusual metals as carcinogens

A review of the literature on unusual metals as carcinogens was carried out. The metals covered are some of the rare earths, copper, silver, gold, mercury, germanium, tin, antimony, lead, platinum, palladium, aluminum, titanium, niobium, manganese, scandium, yttrium, indium, rhodium, and gallium.     

Neutrino intergalactic communication,metal life,and viruses: Part 1 quo vadis ex machina

At one spectrum extreme, Astrobiology conjectures that for exoplanets with Goldilocks conditions, terrestrial-like life is inevitable. Moreover, it is envisaged that via panspermia, terrestrial-like life and its precursors are transferred among galaxies, stars, and within solar systems via transiting comets, asteroids, and planetoids. In addition, expelled stars, which have solar systems, it is inferred, transfer life as well. However, at the other extreme, we propose a paradigm shift that on some planets, subject to non- Goldilocks conditions, metal machine life could arise, ab initio, and evolve viruses, intelligence, and civilizations, conjointly. Accordingly, intelligent mechanized civilizations could readily and efficiently commence space exploration. Furthermore, as a counter paradigm shift, such civilizations could experiment and produce non-metallic life, based on carbon and other non-metal elements, under suitable conditions, related to Goldilocks life. Even a single example of validated interstellar or intergalactic communication received on the Earth would support the existence of life elsewhere. However, the communication platform should not be restricted to electromagnetic radiation. Other platforms should be included as well - one such example, which would require sophisticated technology, is neutrino communication. This is the case for any advanced civilization, be it metal-machine based, biological-based, and carbon-based. In sum, civilizations based on machine life, would be highly productive due to the longevity and hardiness of machine life. However, significant caveats are raised in this brief report, because possibly dissimilar psychologies and intelligence may lead to conflicts between metal machine life and biological life, inter-paradigm conflict.     

Nicotinic Agonists, Antagonists, and Modulators From Natural Sources

1. Acetylcholine receptors were initially defined as nicotinic or muscarinic, based on selective activation by two natural products, nicotine and muscarine. Several further nicotinic agonists have been discovered from natural sources, including cytisine, anatoxin, ferruginine, anabaseine, epibatidine, and epiquinamide. These have provided lead structures for the design of a wide range of synthetic agents.2. Natural sources have also provided competitive nicotinic antagonists, such as the Erythrina alkaloids, the tubocurarines, and methyllycaconitine. Noncompetitive antagonists, such as the histrionicotoxins, various izidines, decahydroquinolines, spiropyrrolizidine oximes, pseudophrynamines, ibogaine, strychnine, cocaine, and sparteine have come from natural sources. Finally, galanthamine, codeine, and ivermectin represent positive modulators of nicotinic function, derived from natural sources.3. Clearly, research on acetylcholine receptors and functions has been dependent on key natural products and the synthetic agents that they inspired.     

Systematics and phylogeny of the caryophyllidia-bearing dorids (Mollusca, Nudibranchia), with descriptions of a new genus and four new species from Indo-Pacific deep waters

The phylogenetic relationships of the caryophyllidia-bearing dorids are studied, based on the examination of the type species of all the genera previously described. The phylogenetic hypothesis supports that the caryophyllidia-bearing dorids are a monophyletic group and the sister group of the clade formed by Astemnotus Ehrenberg, 1831 and Halgerda Bergh, 1880. Several genera previously considered as valid or regarded as uncertain are here synonymized: Peronodoris Bergh, 1904, Trippa Bergh, 1877, Phlegmodoris Bergh, 1878, Petelodoris Bergh, 1881, Kentrodoris Bergh, 1876, Audura Bergh, 1878, Centrodoris P. Fischer, 1883, Anisodoris Bergh, 1898, Awuka Er. Marcus, 1955, Rhabdochiia P. Fischer, 1883, Boreodoris Odhner, 1939, Dictyodoris Bergh, 1880, Gravieria Vayssiere, 1912, Aporodoris Ihering, 1886. The following genera are regarded as valid: Astemnotus, Atagema J.E. Gray, 1850, Jorunna Bergh, 1876, Platydoris Bergh, 1877, Diaulula Bergh, 1878, Rostanga Bergh, 1879, Halgerda Bergh, 1880, Baptodoris Bergh, 1884, Gargamella Bergh, 1894, Alloiodoris Bergh, 1904, Sclerodoris Eliot, 1904, Taringa Er. Marcus, 1955, Thorybopus Bouchet, 1977. The new genus Nophodoris is described based on two new species from New Caledonia deep waters. Two additional new species from New Caledonia belonging to the genera Atagema and Gargamella are also described. Nomenclatural and taxonomic problems are discussed, and several type species, neotypes and lectotypes are selected.     

Molecular phylogeny of the gobioid fishes (Teleostei: Perciformes: Gobioidei)

The phylogeny of groups within Gobioidei is examined with molecular sequence data. Gobioidei is a speciose, morphologically diverse group of teleost fishes, most of which are small, benthic, and marine. Efforts to hypothesize relationships among the gobioid groups have been hampered by the prevalence of reductive evolution among goby species; such reduction can make identification of informative morphological characters particularly difficult. Gobies have been variously grouped into two to nine families, several with included subfamilies, but most existing taxonomies are not phylogenetic and few cladistic hypotheses of relationships among goby groups have been advanced. In this study, representatives of eight of the nine gobioid familes (Eleotridae, Odontobutidae, Xenisthmidae, Gobiidae, Kraemeriidae, Schindleriidae, Microdesmidae, and Ptereleotridae), selected to sample broadly from the range of goby diversity, were examined. Complete sequence from the mitochondrial ND1, ND2, and COI genes (3573 bp) was used in a cladistic parsimony analysis to hypothesize relationships among the gobioid groups. A single most parsimonious topology was obtained, with decay indices indicating strong support for most nodes. Major phylogenetic conclusions include that Xenisthmidae is part of Eleotridae, and Eleotridae is paraphyletic with respect to a clade composed of Gobiidae, Microdesmidae, Ptereleotridae, Kraemeriidae, and Schindleriidae. Within this five-family clade, two clades are recovered. One includes Gobionellinae, which is paraphyletic with respect to Kraemeriidae, Sicydiinae, Oxudercinae, and Amblyopinae. The other contains Gobiinae, also paraphyletic, and including Microdesmidae, Ptereleotridae, and Schindleriidae. Previous morphological evidence for goby groupings is discussed; the phylogenetic hypothesis indicates that the morphological reduction observed in many goby species has been derived several times independently.     

Foliar nutrient levels of broad-leaved tropical trees: A tabular review

Foliar nutrient levels (N, P, S, K, Ca, Mg, Al, Fe, Mn, Zn, Cu, Mo, B), of about 40 tropical and subtropical broad-leaved trees, are listed and classified as deficient, low, intermediate, high, and toxic in each case according to the interpretation and evaluation of the reviewed author(s).     

Oxypred： Prediction and Classification of Oxygen-Binding Proteins

This study describes a method for predicting and classifying oxygen-binding pro- teins. Firstly, support vector machine (SVM) modules were developed using amino acid composition and dipeptide composition for predicting oxygen-binding pro- teins, and achieved maximum accuracy of 85.5% and 87.8%, respectively. Sec- ondly, an SVM module was developed based on amino acid composition, classify- ing the predicted oxygen-binding proteins into six classes with accuracy of 95.8%, 97.5%, 97.5%, 96.9%, 99.4%, and 96.0% for erythrocruorin, hemerythrin, hemo- cyanin, hemoglobin, leghemoglobin, and myoglobin proteins, respectively. Finally, an SVM module was developed using dipeptide composition for classifying the oxygen-binding proteins, and achieved maximum accuracy of 96.1%, 98.7%, 98.7%, 85.6%, 99.6%, and 93.3% for the above six classes, respectively. All modules were trained and tested by five-fold cross validation. Based on the above approach, a web server Oxypred was developed for predicting and classifying oxygen-binding proteins(available from http://www.imtech.res.in/raghava/oxypred/).     

Distribution of N,P, K,Ca, Mg,S, Zn,Fe, Mn and Cu in groundnut

Summary Concentration of N, P, K, Ca, Mg and S in summer groundnut crop was higher than in kharif while Zn, Fe, Mn and Cu contents were higher in summer crop. Kernel's N, P and Zn; Leaflet's Ca and Mn; Stem's K and Fe; Root's S and Cu and Petiole's Mg contents were highest. Shell's N, P, K, Mg, S, Zn and Cu; Kernel's Ca, Fe and Mn contents were the least. N, P, K, S, Zn and Cu concentrations decreased linearly as the crop grew. Ca, Mg, Fe and Mn concentrations did not display any distinct pattern. Ca concentration was positively correlated with pod yield in both the seasons.