The maize autonomous element Activator (Ac) shows a minimal germinal excision frequency of 0.2%–0.5% in transgenic Arabidopsis thaliana plants

Summary The autonomous mobile element Activator from Zea mays was introduced into Arabidopsis thaliana via Agrobacterium-mediated gene transfer. The use of a chimaeric construct, where the Ac element is located in the leader of the neomycin phosphotransferase (NPT II) gene, enabled the excision of Ac to be monitored by assaying for the reconstitution of NPT II gene activity. Using this approach, the transpositional activity of AC was initially studied in primary transformants. About 50% of the regenerating Ac transformants showed evidence for excision of the element. Reintegration of Ac was confirmed by Southern blot analysis. Transposition events are transmitted to the F1 generation with a minimal frequency of 0.3%. In a few exceptional cases they are detected in a high proportion of the F1 generation. Seedlings from the F2 and F3 generations were assayed for the rate of germinal excisions by scoring for kanamycin resistance. The minimal frequency of germinal excision events amounts to 0.2%–0.5% and hence allows the use of the Ac element for gene tagging purposes in A. thaliana.     

Cellular redox poise modulation; the role of coenzyme Q10, gene and metabolic regulation

In this communication, the concept is developed that coenzyme Q₁₀ has a toti-potent role in the regulation of cellular metabolism. The redox function of coenzyme Q₁₀ leads to a number of outcomes with major impacts on sub-cellular metabolism and gene regulation. Coenzyme Q₁₀'s regulatory activities are achieved in part, through the agency of its localization in the various sub-cellular membrane compartments. Its fluctuating redox poise within these membranes reflects the cell's metabolic micro-environments. As an integral part of this process, H₂O₂ is generated as a product of the normal electron transport systems to function as a mitogenic second messenger informing the nuclear and mitochondrial (chloroplast) genomes on a real-time basis of the status of the sub-cellular metabolic micro-environments and the needs of that cell. Coenzyme Q₁₀ plays a major role both in energy conservation, and energy dissipation as a component of the uncoupler protein family. Coenzyme Q₁₀ is both an anti-oxidant and a pro-oxidant and of the two the latter is proposed as its more important cellular function. Coenzyme Q₁₀ has been reported, to be of therapeutic benefit in the treatment of a wide range of age related degenerative systemic diseases and mitochondrial disease. Our over-arching hypotheses on the central role played by coenzyme Q₁₀ in redox poise changes, the generation of H₂O₂, consequent gene regulation and metabolic flux control may account for the wide ranging therapeutic benefits attributed to coenzyme Q₁₀.     

Mapping of genes on the linear chromosome of the bacterium Borrelia burgdorferi: Possible locations for its origin of replication

Molecular clones of Borrelia burgdorferi, aetiologic agent of Lyme borreliosis, were isolated and analysed by DNA sequence determination. This procedure yielded B. burgdorferi homologues of gidA, gyrB, gyrA, ftsA and ftsZ. The genes were located on the physical map of the B. burgdorferi linear chromosome. Also mapped were the genes fla and p60 while dnaA was mapped using a heterologous probe. gyrA and gyrB were found to be in tandem and were mapped, along with dnaA at the centre of the chromosome. gidA was located close to the left hand extremity of the chromosome. Because gyrB, dnaA and gidA are normally located within 50 kb of the origin of replication (oriC), we propose two possible sites for oriC in the B. burgdorferi linear chromosome.     

EB病毒融合基因重组BCG穿梭质粒的构建及表达

分别以卡介苗(BCG)和EB病毒融合基因cDNA为模板,通过PCR扩增得到139bp的BCG-Ag85B信号肽序列和2291bp的Z2A基因序列。将BCG-Ag85B信号肽序列与大肠杆菌-卡介苗穿梭表达载体pMV261重组,得到重组质粒pMVS。再将EB病毒融合基因序列Z2A亚克隆至pMVS中,得到重组质粒pMVZ2A,电转化导入BCG。SDS-PAGE分析结果表明,构建的重组质粒pMVZ2A经双酶切、PCR扩增及测序鉴定证实,克隆基因BCG-Ag85B信号肽和Z2A正确插入载体pMV261,电转化导入BCG,能够在BCG中分泌表达。     

日本大耳白黑眼兔在不同生长阶段蛋白质营养与肝脏相关基因表达的关系

目的研究日粮粗蛋白水平和生长阶段对日本大耳白黑眼兔（WHBE兔）肝脏相关基因表达的影响。方法采用两因素实验设计,分别选取断奶和2月龄WHBE兔各20只进行为期1个月的幼兔期和育成兔期饲养实验,每个阶段实验均将兔随机分为5组,各组日粮粗蛋白水平分别为12%、14%、16%、18%和20%,实验结束,用实时荧光定量PCR技术测定肝脏胰岛素样生长因子-1（IGF-1）和磷酸烯醇式丙酮酸羧激酶-C（PEPCK-C）的mRNA的表达丰度。结果生长阶段和粗蛋白水平均对WHBE兔肝脏IGF-1mRNA的表达丰度有显著影响,育成兔期IGF-1 mRNA的表达丰度明显高于幼兔期;日粮粗蛋白水平为16%和18%时IGF-1mRNA表达丰度较高,显著高于其他3组。PEPCK-C mRNA的表达也以16%粗蛋白组最高,幼兔期和育成兔期之间差异无显著性。结论日粮粗蛋白水平对IGF-1和PEPCK-C基因表达有显著影响,生长阶段与IGF-1mRNA表达密切相关。     

新型PEI衍生物的体外表征及对巨噬细胞的转染效果研究

目的:以PEI1.8kDa为基础连接戊二醛合成新型可降解PEI衍生物GPEI后,研究GPEI包裹质粒转染巨噬细胞后对巨噬细胞合成蛋白及表型的影响。方法:合成GPEI后,检测了GPEI质粒聚合物的粒径及电位,并通过透射电镜观察聚合物形态,通过免疫荧光、WB、RT-PCR、Elisa及Transwell实验检测巨噬细胞RNA、蛋白、终产物水平变化及表型改变。结果:GPEI能够包裹质粒,形成100 nm左右带有正电荷的聚合物,WB、RT-PCR结果表明GPEI转染较PEI25kDa有优势(P0.05),巨噬细胞转染后6天持续分泌PGI2(P0.05)。结论:以PEI1.8kDa为基础合成的GPEI能够包裹PTGIS质粒并将其转染进巨噬细胞后对巨噬细胞的表型有明显影响。     

番茄的耐盐性与耐盐转基因番茄

由于土壤盐渍化的日益加剧导致作物大量减产,通过基因工程的方法将耐盐基因转入作物,培育耐盐作物新品种是开发利用盐碱地的一种经济、有效的手段.番茄是一种栽培广泛,具有重要经济价值的蔬菜,又是基因工程的模式生物之一,开展番茄耐盐性研究具有重要意义.分析了番茄的耐盐性和转耐盐基因番茄的研究现状.     

靶向整合研究进展

基因治疗的目的是将遗传物质导入细胞并使之得到适宜水平的表达,以纠正机体的遗传缺陷,恢复细胞的正常功能或杀死癌细胞及致病微生物。目前广泛应用的病毒及非病毒载体不能很好地满足临床要求,是基因治疗亟需解决的关键技术。同源重组介导的基因靶向性整合,是遗传性疾病基因治疗的较佳方案。近年来有关同源重组研究的进展,使得其应用于基因治疗成为可能。     

Limiting inbreeding in disjunct and isolated populations of a woody shrub

Pollen movements and mating patterns are key features that influence population genetic structure. When gene flow is low, small populations are prone to increased genetic drift and inbreeding, but naturally disjunct species may have features that reduce inbreeding and contribute to their persistence despite genetic isolation. Using microsatellite loci, we investigated outcrossing levels, family mating parameters, pollen dispersal, and spatial genetic structure in three populations of Hakea oldfieldii, a fire‐sensitive shrub with naturally disjunct, isolated populations prone to reduction in size and extinction following fires. We mapped and genotyped a sample of 102 plants from a large population, and all plants from two smaller populations (28 and 20 individuals), and genotyped 158–210 progeny from each population. We found high outcrossing despite the possibility of geitonogamous pollination, small amounts of biparental inbreeding, a limited number of successful pollen parents within populations, and significant correlated paternity. The number of pollen parents for each seed parent was moderate. There was low but significant spatial genetic structure up to 10 m around plants, but the majority of successful pollen came from outside this area including substantial proportions from distant plants within populations. Seed production varied among seven populations investigated but was not correlated with census population size. We suggest there may be a mechanism to prevent self‐pollination in H. oldfieldii and that high outcrossing and pollen dispersal within populations would promote genetic diversity among the relatively small amount of seed stored in the canopy. These features of the mating system would contribute to the persistence of genetically isolated populations prone to fluctuations in size.