体外培养瘤胃微生物总DNA的提取与纯化

主要目的是寻找一种能最大限度获取所有类型的瘤胃微生物大片段DNA的提取方法,为后期分子生物学技术研究瘤胃微生物奠定基础。采用液氮冻融+溶菌酶+CTAB和SDS相结合的方法处理瘤胃样品并充分破壁,再采用酚/氯仿/异戊醇抽提、异丙醇沉淀获得DNA粗提物,经过试剂盒纯化后得到瘤胃微生物DNA样品。经特异性引物PCR检测,本方法所提取的DNA包含细菌、古细菌、真菌及原虫四类微生物信息。因此,采用该方法提取的DNA可用于后期实时定量PCR或DGGE等研究。     

生物DNA图谱中干扰下的特征识别与分割

生物DNA图谱的拍摄容易受到仪器、周围环境等各种因素的干扰,当前方法的生物DNA图谱识别效果不佳,提出一种改进的生物DNA图谱识别方法,根据采集得到的DNA图谱结构特征,将生物DNA图谱输入多层迭代分割网络中,采集输入生物DNA图谱的局部特征,对采集到的特征进行层层迭代处理,得到更高级特征,利用卷积能够区分细小特征这一优势,将最终获取的特征转换成卷积操作,将该结果看作是输入生物DNA图谱的表征,可有效去除粘黏干扰,依据该特征进行分类,实现生物DNA图谱识别。实验结果表明,采用所提方法对生物DNA图谱进行识别,能够在很大程度上提高生物DNA图谱识别的准确性。     

Ribonucleotides in DNA newly synthesised in 3T6 cells in vivo.

Within the field of DNA replication, considerable interest has focused in recent years on the mechanism of initiation of synthesis of DNA molecules. In vitro replication systems from Escherichia coli have been instrumental in uncovering a priming function fo9r ribonucleotides on the earliest intermediates of DNA polymerisation in vitro and in identifying the proteins involved. In vitro replication systems from mammalian cells that permit the use of the phosphate-transfer method for detection of RNA-DNA junctions as well as direct labelling of the RNA moiety of the molecules have suggested a similar role for ribonucleotides in DNA synthesis in eukaryotes. However, the existence of this mechanism in mammalian cells in vivo has not been established. Here we report the first evidence that a significant proportion of the earliest intermediates in mammalian DNA polymerisation in vivo do, in fact, possess ribonucleotides, presumably because their synthesis was initiated with one or more ribonucleotides.     

DNA in a material world

The specific bonding of DNA base pairs provides the chemical foundation for genetics. This powerful molecular recognition system can be used in nanotechnology to direct the assembly of highly structured materials with specific nanoscale features, as well as in DNA computation to process complex information. The exploitation of DNA for material purposes presents a new chapter in the history of the molecule.     

DNA甲基化研究进展

DNA甲基化已成为阐明正常和病理基因表达现象的重要机制,因而已成为当前分子生物学的研究热点之一。对DNA甲基化的转录抑制机制、在肿瘤发生中的作用、与细胞衰老和凋亡的关系、抑制剂以及分析方法等方面的研究新进展进行了综述。     

三丁基锡对褐菖鲉肝脏DNA损伤的研究

利用碱解旋法研究了三丁基锡(TBT)暴露对鱼肝脏DNA造成的单链断裂损伤.结果表明:0.5、1、5和10mg/kg(Bm)TBT腹腔注射7d后,褐菖鲉(Sebastiscus marmoratus)肝脏DNA的损伤程度存在剂量依赖关系,随着注射剂量的增加而增加.环境相关浓度(1、10、100ng/L含Sn量)的TBT通过水体对褐菖鲉进行暴露,肝脏DNA的损伤程度,总体上随着暴露时间的延长和暴露浓度的增大而增加.本研究为鱼类肝脏DNA损伤作为生物标志物来指示水体TBT污染的有效性提供了依据.     

Linearly programmed DNA-based molecular computer operated on magnetic particle surface in test-tube

The postgenomic era has seen an emergence of new applications of DNA manipulation technologies, including DNA-based molecular computing. Surface DNA computing has already been reported in a number of studies that,however, all employ different mechanisms other than automaton functions. Here we describe a programmable DNA surface-computing device as a Turing machine-like finite automaton. The laboratory automaton is primarily composed of DNA (inputs, output-detectors, transition molecules as software), DNA manipulating enzymes and buffer system that solve artificial computational problems autonomously. When fluoresceins were labeled in the 5‘ end of (-) strand of the input molecule, direct observation of all reaction intermediates along the time scale was made so that the dynamic process of DNA computing could be conveniently visualized. The features of this study are: (i) achievement of finite automaton functions by linearly programmed DNA computer operated on magnetic particle surface and (ii)direct detection of all DNA computing intermediates by capiilary electrophoresis. Since DNA computing has the massive parallelism and feasibility for automation, this achievement sets a basis for large-scale implications of DNA computing for functional genomics in the near future.     

随机DNA序列中ORF的分布

研究了组分约束下的随机 DNA序列中 ORF数目、ORF的长度与随机序列总长度和GC含量之间的关系 .结果表明 ,ORF数目的对数与 ORF的长度之间存在很好的线性关系 ;ORF的最大长度随序列长度的增加而变长 ,ORF的总数与序列长度成正比 ;ORF的总数目随着 GC含量的增加明显减少 ;随着 GC含量的增加 ,长度较短的 ORF数目相对减少 ,长度较长的 ORF数目相对增加 ;随着序列长度的增加 ,长度较短的 ORF相对增多 .     

Reassembly of shattered chromosomes in Deinococcus radiodurans

Dehydration or desiccation is one of the most frequent and severe challenges to living cells. The bacterium Deinococcus radiodurans is the best known extremophile among the few organisms that can survive extremely high exposures to desiccation and ionizing radiation, which shatter its genome into hundreds of short DNA fragments. Remarkably, these fragments are readily reassembled into a functional 3.28-megabase genome. Here we describe the relevant two-stage DNA repair process, which involves a previously unknown molecular mechanism for fragment reassembly called 'extended synthesis-dependent strand annealing' (ESDSA), followed and completed by crossovers. At least two genome copies and random DNA breakage are requirements for effective ESDSA. In ESDSA, chromosomal fragments with overlapping homologies are used both as primers and as templates for massive synthesis of complementary single strands, as occurs in a single-round multiplex polymerase chain reaction. This synthesis depends on DNA polymerase I and incorporates more nucleotides than does normal replication in intact cells. Newly synthesized complementary single-stranded extensions become 'sticky ends' that anneal with high precision, joining together contiguous DNA fragments into long, linear, double-stranded intermediates. These intermediates require RecA-dependent crossovers to mature into circular chromosomes that comprise double-stranded patchworks of numerous DNA blocks synthesized before radiation, connected by DNA blocks synthesized after radiation.     

Pitfalls in the analysis of ancient human mtDNA

The retrieval of DNA from ancient human specimens is not always successful owing to DNA deterioration and contamination although it is vital to provide new insights into the genetic structure of ancient people and to reconstruct the past history. Normally, only short DNA fragments can be retrieved from the ancient specimens. How to identify the authenticity of DNA obtained and to uncover the information it contained are difficult. We employed the ancient mtDNAs reported from Central Asia (including Xinjiang, China) as an example to discern potentially extraneous DNA contamination based on the updated mtDNA phylogeny derived from mtDNA control region, coding region, as well as complete sequence information. Our results demonstrated that many mtDNAs reported are more or less problematic.Startim, from a reliable mtDNA phylogeney and combining the available modern data into analysis, one can ascertain the authenticity of the ancient DNA, distinguish the potential errors in a data set, and efficiently decipher the meager information it harbored. The reappraisal of the mtDNAs with the age of more than 2000 years from Central Asia gave support to the suggestion of extensively (pre)historical gene admixture in this region.     

DNA sequences of telomeres maintained in yeast

Telomeres, the ends of eukaryotic chromosomes, have long been recognized as specialized structures. Their stability compared with broken ends of chromosomes suggested that they have properties which protect them from fusion, degradation or recombination. Furthermore, a linear DNA molecule such as that of a eukaryotic chromosome must have a structure at its ends which allows its complete replication, as no known DNA polymerase can initiate synthesis without a primer. At the ends of the relatively short, multi-copy linear DNA molecules found naturally in the nuclei of several lower eukaryotes, there are simple tandemly repeated sequences with, in the cases analysed, a specific array of single-strand breaks, on both DNA strands, in the distal portion of the block of repeats. In general, however, direct analysis of chromosomal termini presents problems because of their very low abundance in nuclei. To circumvent this problem, we have previously cloned a chromosomal telomere of the yeast Saccharomyces cerevisiae on a linear DNA vector molecule. Here we show that yeast chromosomal telomeres terminate in a DNA sequence consisting of tandem irregular repeats of the general form C1-3A. The same repeat units are added to the ends of Tetrahymena telomeres, in an apparently non-template-directed manner, during their replication on linear plasmids in yeast. Such DNA addition may have a fundamental role in telomere replication.     

Altered DNA ligase I activity in Bloom's syndrome cells

Cells from patients with Bloom's syndrome, a rare disease associated with increased cancer frequency, exhibit cytological abnormalities. These include increased numbers of homologous chromatid interchange figures and sister-chromatid exchanges, together with abnormally slow replicon-fork progression and retarded rate of DNA-chain maturation, and suggest that the primary defect in this recessive disorder affects S-phase DNA replication. DNA ligases and DNA polymerases have long been prime candidates for abnormality in Bloom's syndrome, but various studies of DNA polymerases in Bloom's syndrome cells have disclosed no abnormalities. Evidence is presented here, as in the accompanying paper from a different laboratory, for the existence in Bloom's syndrome of an abnormality of the DNA ligase involved in semi-conservative DNA replication.     

Thiobase DNA: the chemistry and some applications in cancer studies

This article reviews the chemical methods for preparation of base-modified DNA with a particular focus on thiobases. Thiobases have certain unique properties, such as easy alkylation, ready oxidation and strong absorption in the near UV longer wavelength. These chemical and physical properties are retained in DNA containing thiobase and are useful for chemical manipulation and photo crosslinking of these modified DNA with other biomolecules of interest. The ability to make DNA containing thiobases or their functionalized analogues has added new research tools for DNA repair studies, and it is believed that such modified DNA can be of great use in many biological studies.     

MDC1 is coupled to activated CHK2 in mammalian DNA damage response pathways

Forkhead-homology-associated (FHA) domains function as protein-protein modules that recognize phosphorylated serine/threonine motifs. Interactions between FHA domains and phosphorylated proteins are thought to have essential roles in the transduction of DNA damage signals; however, it is unclear how FHA-domain-containing proteins participate in mammalian DNA damage responses. Here we report that a FHA-domain-containing protein-mediator of DNA damage checkpoint protein 1 (MDC1; previously known as KIAA0170)--is involved in DNA damage responses. MDC1 localizes to sites of DNA breaks and associates with CHK2 after DNA damage. This association is mediated by the MDC1 FHA domain and the phosphorylated Thr 68 of CHK2. Furthermore, MDC1 is phosphorylated in an ATM/CHK2-dependent manner after DNA damage, suggesting that MDC1 may function in the ATM-CHK2 pathway. Consistent with this hypothesis, suppression of MDC1 expression results in defective S-phase checkpoint and reduced apoptosis in response to DNA damage, which can be restored by the expression of wild-type MDC1 but not MDC1 with a deleted FHA domain. Suppression of MDC1 expression results in decreased p53 stabilization in response to DNA damage. These results suggest that MDC1 is recruited through its FHA domain to the activated CHK2, and has a critical role in CHK2-mediated DNA damage responses.     

Newly replicated DNA is associated with DNA topoisomerase II in cultured rat prostatic adenocarcinoma cells

DNA topoisomerases have been proposed to function in a variety of genetic processes in both prokaryotes and eukaryotes. Here, we have assessed the role of DNA topoisomerase II in mammalian DNA replication by determining the proximity of newly synthesized DNA to covalent enzyme-DNA complexes generated by treating cultured rat prostatic adenocarcinoma cells with teniposide. Teniposide (VM-26), an epipodophyllotoxin, is known to interact with mammalian DNA topoisomerase II so as to trap the enzyme in a covalent complex with DNA. We have found that the teniposide-induced trapping of such complexes requires MgCl2, is stimulated by ATP and is inhibited by novobiocin. The formation of covalent complexes seems to be reversible on removal of teniposide. Furthermore, analysis of the covalent complexes formed between 3H-thymidine pulse-labelled DNA and topoisomerase II following teniposide treatment reveals a direct association of the enzyme with nascent DNA fragments. Our results suggest that DNA topoisomerase II may interact with newly replicated daughter DNA molecules near DNA replication forks in mammalian cells.     

Recombination occurs during telomere formation in yeast

Short stretches of cloned telomeric sequences are necessary and sufficient for telomere formation in yeast as long as the sequences are present in the same orientation as they are found in vivo. During telomere formation, DNA termini usually undergo RAD52-independent recombination with other DNA termini as would be predicted by models of recombination-mediated telomere replication.     

高等植物中的DNA解旋酶

综述了在离体实验条件下高等植物DNA解旋酶在以下多方面具有重要的生物学功能：包括DNA重组、DNA复制、翻译启始、rDNA转录及在pre-rRNA加工过程早期阶段，双链断裂修复、端粒长度维持、核苷酸切除修复、花发育中的细胞分裂／增殖、基因组甲基化方式保持、植物细胞周期和细胞基本生命活动的维持。最近玉米基因组中发现的解旋子(helitron)插入说明高等植物DNA解旋酶可能在植物生长与发育中有重要调控作用。因而有着重要的生物技术应用价值。     

高等植物中的DNA解旋酶

综述了在离体实验条件下高等植物DNA解旋酶在以下多方面具有重要的生物学功能：包括DNA重组、DNA复制、翻译启始、rDNA转录及在prerRNA加工过程早期阶段,双链断裂修复、端粒长度维持、核苷酸切除修复、花发育中的细胞分裂/增殖、基因组甲基化方式保持、植物细胞周期和细胞基本生命活动的维持。最近玉米基因组中发现的解旋子（helitron)插入说明高等植物DNA解旋酶可能在植物生长与发育中有重要调控作用。因而有着重要的生物技术应用价值。     

柳树RAPD反应体系均匀设计试验研究

从柳树幼苗的嫩叶中提取基因组DNA作为模板,运用均匀设计方法,获得;柳树RAPD扩增反应的优化体系：25μL反应体系中,DNA模板用量70 ng,引物浓度0.5mmol.L-1,Mg2＋浓度3mmol.L-1,TaqDNA聚合酶用量1.0 U,dNTP浓度0.3mmol.L-1.用16条引物进行验证,证实该体系重复性好、结果稳定.