扩增片断长度多态性(AFLP)荧光标记和银染技术的比较分析

实验采用AFLP的荧光标记分析技术和常规的AFLP银染技术,对施用农药和未施用农药的狼蛛基因多态性进行分析.用4对引物扩增和银染法共检测出32条带,荧光标记技术共检测出223条带.荧光技术比银染方法在每个位点上多检测到24条带,检测效果更为理想,更适于进行遗传多样性分析和研究;对两种方法的费用和工作效率做了初步分析,AFLP荧光标记技术的检测效率是银染法的690倍,同时对AFLP荧光标记技术中低成本、高通量多重PCR体系的建立及Genescan软件数据分析中出现的一些具体问题进行了探讨.     

顽拗植物龙眼基因组DNA提取方法的研究

为从顽拗植物龙眼(Dimocarpus longan Lour.)叶片中获得可供后续分子生物学操作的基因组DNA．针对其组织细胞内富含多酚、多糖、单宁及色素等物质的特点,采用改进的CTAB法和SDS法,即在核裂解之前先破碎细胞．将存在于细胞质中的次生物质去除后再裂解细胞桉．结合其它一些改进措施．提取到的DNA沉淀呈纯白色．极易溶解于TE中。两种改进方法的OD260和OD280比值分别达到1.82和1．73,其鲜叶基因组DNA产量分别为103ug/g和127ug／g：RAPD扩增条带清晰,丰富．完全满足后续分子生物学操作的要求,其中改良CTAB方法效果更为理想,与之埘比．传统的CTAB法和SDS法提取到的DNA沉淀呈浅黄色甚至红褐色,很难被TE溶解,其OD260和OD280比值均低于1．5,也得不到扩增产物。     

PCP degradation is mediated by closely related strains of the genus Sphingomonas

There have been numerous reports in the literature of diverse bacteria capable of degrading pentachlorophenol (PCP). In order to gain further insight into the phylogenetic relationships of PCP-degrading bacteria, we examined four strains: Arthrobacter sp. strain ATCC 33790, Flavobacterium sp. strain ATCC 39723, Pseudomonas sp. strain SR3, and Sphingomonas sp. strain RA2. These organisms were isolated from different geographical locations and all of them degrade high concentrations (100–200 mg/L) of PCP. Southern blot analyses determined that these bacteria all harbour DNA that encodes similar, if not identical, genes involved in PCP degradation. Comparison of the 16S rRNA nucleotide sequences revealed that these organisms were very closely related and, in fact, represent a monophyletic group. The 16S rRNA analyses together with fatty acid and sphingolipid analyses strongly suggest that the four strains are members of the genus Sphingomonas . The close relationship of the four organisms is supported by nucleotide sequence analysis data of the pcpB locus encoding PCP-4-monooxygenase, the first enzyme in the PCP degradative pathway.     

大规模生产制备质粒DNA技术进展

质粒DNA疫苗或基因治疗剂是生物制品的新品种。鉴于质粒DNA表达效率低、持续时间短,需建立一套大规模生产制备质粒DNA的工艺,即发酵、碱性裂解、分离纯化及质量控制。目前质粒DNA大规模生产已经达到千克级水平,一些产品正在进行临床实验。     

Enzymes of Direct,Excision, and Mismatch DNA Repair in Pro- and Eukaryotes and Their Biological Role

DNA repair is of paramount importance to protect the integrity of the cell genome. Over the last decade, DNA repair enzymes have been intensely studied owing to their role in mutagenesis, carcinogenesis, and aging. This led to discoveries of novel enzymes, protein factors as well as their intermolecular complexes involved in the repair process. Alternative pathways of cell response to the same type of damage were identified. Data on the tertiary structure of a number of enzymes and mechanisms of their molecular interaction were obtained. This paper is an overview of latest advances in the research of cell response to DNA damage via direct, base and nucleotide excision, and mismatch DNA repair.     

HCV编码病毒结构蛋白的DNA免疫研究

应用HCVC (pC)和HCVCE1E2 (pCE1E2 )重组体转染真核细胞并且通过肌注免疫BALB/C小鼠 ,对其体液免疫和细胞免疫进行检测。所用的 pC和 pCE1E2 均可在真核细胞内表达出特异性HCVC蛋白 ;肌注DNA免疫后均可诱导出BALB/C小鼠的体液和细胞免疫反应 ,抗体反应的A值 :pC组为 0 .358± 0 .0 96 ,pCE1E2 组为 0 .4 15± 0 .12 7;CTL活力 pC组为 18.6 5%± 5.72 % ,pCE1E2 组为 2 0 .0 7%± 11.11% ;通过免疫鼠荷瘤检测体内CTL反应 ,可观察到免疫组鼠发瘤时间滞后 ,发瘤部位减少和存活时间延长。在开发和研制HCV疫苗的过程中 ,DNA免疫是在快速构建、评价和筛选免疫原方面的有效办法。     

Structural and Functional Characterisation of a Conserved Archaeal RadA Paralog with Antirecombinase Activity

DNA recombinases (RecA in bacteria, Rad51 in eukarya and RadA in archaea) catalyse strand exchange between homologous DNA molecules, the central reaction of homologous recombination, and are among the most conserved DNA repair proteins known. RecA is the sole protein responsible for this reaction in bacteria, whereas there are several Rad51 paralogs that cooperate to catalyse strand exchange in eukaryotes. All archaea have at least one (and as many as four) RadA paralog, but their function remains unclear. Herein, we show that the three RadA paralogs encoded by the Sulfolobus solfataricus genome are expressed under normal growth conditions and are not UV inducible. We demonstrate that one of these proteins, Sso2452, which is representative of the large archaeal RadC subfamily of archaeal RadA paralogs, functions as an ATPase that binds tightly to single-stranded DNA. However, Sso2452 is not an active recombinase in vitro and inhibits D-loop formation by RadA. We present the high-resolution crystal structure of Sso2452, which reveals key structural differences from the canonical RecA family recombinases that may explain its functional properties. The possible roles of the archaeal RadA paralogs in vivo are discussed.     

Regulation of the DNA Damage Response to DSBs by Post-Translational Modifications

Damage to the genetic material can affect cellular function in many ways. Therefore, maintenance of the genetic integrity is of primary importance for all cells. Upon DNA damage, cells respond immediately with proliferation arrest and repair of the lesion or apoptosis. All these consequences require recognition of the lesion and transduction of the information to effector systems. The accomplishment of DNA repair, but also of cell cycle arrest and apoptosis furthermore requires protein-protein interactions and the formation of larger protein complexes. More recent research shows that the formation of many of these aggregates depends on post-translational modifications. In this article, we have summarized the different cellular events in response to a DNA double strand break, the most severe lesion of the DNA.     

Specific sensing of poly G by the aluminum-salophen complex

The Al(III)-salophen complex 1 exhibited strong spectroscopic changes specifically upon addition of polyG and GpG, while double stranded DNA and RNA, and single stranded polyA, polyU and polyC induced negligible spectral changes of 1. Titrations with mono-nucleotides yielded no spectroscopic changes, revealing that there must be at least two consecutive guanines in single stranded oligonucleotide structure for a measurable spectroscopic change of 1. Preliminary results show that 1 has moderate antiproliferative effect on a number of human tumour cell lines.