真核生物的基因组织和结构

真核生物的基因组DNA被包装在染色体中。染色体的化学物质是染色质,它是由DNA和核蛋白组成。染色体DNA含有几乎所有真核生物的基因、重复序列和基因间序列。基因是由一段DNA序列组成,有编码序列和非编码序列。真核基因的编码序列是不连续的,它被插入序列分隔,而各真核基因之间则由基因间序列相隔。基因的结构和表达与核内蛋白质有着密切的关系。     

一种有效的重复序列识别算法

重复序列的分析是基因组研究中的一个重要课题,进行这一研究的基础则是从基因组序列中快速有效地找出其中的重复序列。一种投影拼接算法,即利用随机投影获得候选片断集合,利用片断拼接对候选片断进行拼接,以发现基因组中的重复序列。分析了算法的计算复杂度,构造了半仿真测试数据,对算法的测试结果表明了其有效性。     

鲫鱼Hind Ⅲ高重复DNA序列的分子克隆

基因组DNA高重复序列的研究有助于解释许多重要的生命现象 ,如基因调节、基因转座、基因进化等 ,还可以用于进行种群的遗传分析。鱼类的DNA高重复序列研究资料较少 ,曾在鲤科鱼类发现HindⅢ高重复序列家族。本研究用HindⅢ内切酶消化 ,从鲫鱼 (Carassiusauratusauratus)基因组DNA也克隆出一种独特的高重复序列。序列测定揭示该重复序列长度为 175bp ,在单倍体基因组的拷贝数为 1× 10 5。鲫鱼HindⅢ高重复序列与鲫鱼属 (Carassius)其它同类已知的高重复序列存在某种程度的变异 ,而与鲤科其它属的已知的HindⅢ高重复序列完全不同     

中华鲟(Acipenser sinensis)及相关种类的mtDNA控制区串联重复序列及其进化意义

采用 PCR技术和 DNA测序技术 ,发现了我国一级珍稀保护动物中华鲟 ( Acipensersinensis)线粒体 DNA( mt DNA)的控制区 ( D- loop)存在数目不等的串联重复序列 ,该重复序列造成了中华鲟广泛的异质性现象 .从分子水平进行了不同类型重复序列变化规律的研究 ,同时还初探了重复序列在我国其它几种鲟鱼类的存在情况 ,发现在白鲟 ( Psephurus gladius)、达氏鲟 ( A.dabryanus)和史氏鲟 ( A.schrenckii)均存在类似的重复序列结构 .序列比较分析表明 ,不同鲟鱼类重复序列在鲟鱼类进化过程中扮演着一定的角色 ,很有可能碱基差异大小与它们的亲缘关系的远近呈正相关 .     

分子生物学教材中的真核生物基因组重复序列

现行的高校分子生物学教材中主要以重复频率为依据对重复序列进行分类,对于小卫星DNA及微卫星DNA是属于高度或是中度重复序列存在不同见解。提出依据重复频率及空间结构分布两个方面对重复序列进行分类,并建议按照重复频率将小卫星DNA及微卫星DNA归属于中度重复序列。     

基因文库的鉴定和应用

由于草菇是一种同宗结合的食用真菌,这给草菇的杂交育种带来了一定的困难。本文在建立草菇部分基因文库的基础之上,对草菇的基因文库进行了鉴定。在草菇基因文库中任意抽取72个克隆,利用专一的PCR方法,测出在草菇基因文库中,草菇基因组DNA的平均大小为1156个碱基对。在基因文库中任意选择53个克隆,利用专一的PCR进行DNA扩增以及dig非同位素标记,用于和草菇基因组DNA杂交。在测试的53个克隆中有8％的高度重复序列,36％中度重复序列和56％的低度重复序列。     

三核苷酸重复序列失稳定机制:重复序列形成non-B二级结构的必要性和细胞反式作用因子的作用

与三核苷酸重复序列CAG.CTG、CGG·CCG和GAA·TTC扩增和缺失有关的分子机制尚不能得到清楚的阐释.体外研究表明,上述疾病相关的重复序列可以在体外形成non-B二级结构,并介导重复序列扩增.然而,迄今为止,类似的观察尚未在体内研究过程中得以实现.利用模型生物大肠杆菌和酵母等进行的有关研究并不能模拟三核苷酸重复序列的扩增,这暗示三核苷酸重复序列的体内扩增可能与重复序列形成non-B二级结构关联性并不大.尽管理论上较长的三核苷酸重复序列可以在复制和后复制过程中较易形成non-B DNA二级结构,但这样的二级结构倾向于导致重复序列出现"脆性",而不是扩增.事实上,患者所具有的三核苷酸重复序列扩增并非一定需要通过non-B二级结构的介导,这些重复序列的扩增是可以通过一种RNA转录诱导的局部DNA重复序列的复制和其后的DNA重排得以发生.     

一种基于关键路径法的DNA计算用寡核苷酸序列设计算法

在原有的生物大分子序列比对算法的基础上,结合图论中的关健路径法,提出了一种新的计算两寡核苷酸序列间最大配对程度的算法。采用此算法结合生成并测试的方法,能够寻找给定长度的一组适用于DNA计算的寡核苷酸序列。同时采用DNA芯片杂交方法验证了用该算法设计的一组序列的杂交特异性。     

拟南芥DNA探针的比较基因组原位杂交揭示的拟南芥与远缘植物基因组间的同源性

采用生物素标记的拟南芥基因组DNA探针在75%杂交严谨度下对双子叶植物番茄、蚕豆和单子叶植物水稻、玉米、大麦的染色体进行了比较基因组荧光原位杂交（comparative genomic in situ hybridization,cGISH）分析,以揭示拟南芥与远缘植物基因组间的同源性.cGISH信号代表了拟南芥基因组DNA中的重复DNA与靶物种染色体上同源序列的杂交.探针DNA在所有靶物种的全部染色体上都产生了杂交信号.杂交信号为散在分布,并呈现随基因组增大,杂交信号增多,且分布更加分散的趋势.所有靶物种的核仁组织区（NOR）都显示了明显强于其他区域的杂交信号,表明拟南芥基因组DNA探针可用于植物NOR的物理定位.在所有的靶物种中,信号主要分布在染色体的臂中间区和末端,着丝粒或近着丝粒区有少数信号分布.大麦染色体显示了与C-和N-带不同的独特的cGISH信号带型,表明此探针可用于不同植物染色体的识别.这些结果表明,拟南芥基因组与远缘植物基因组之间,除rDNA和端粒重复序列外,还存在其它同源的重复DNA;一些重复DNA序列在被子植物分歧进化为单子叶和双子叶植物之前就已存在,虽经历了长期的进化过程,至今在远缘物种之间仍保持了较高的同源性.结果还提示,大基因组中古老而保守的重复DNA在进化过程中发生了明显的扩增.     

基于DTW距离的DNA序列相似性分析

在DNA序列相似性的研究中,通常采用的动态规划算法对空位罚分函数缺乏理论依据而带有主观性,从而取得不同的结果,本文提出了一种基于DTW（Dynamic Time Warping,动态时间弯曲）距离的DNA序列相似性度量方法可以解决这一问题．通过DNA序列的图形表示把DNA序列转化为时间序列,然后计算DTW距离来度量序列相似度以表征DNA序列属性,得到能够比较DNA序列相似性度量方法,并用这个方法比较分析了七种东亚钳蝎神经毒素（Buthusmartensi Karsch neurotoxin）基因序列的相似性,验证了该度量方法的有效性和准确性．     

Characterisation of a short interspersed repeat (Mermaid) that has family members on human chromosome 21 and elsewhere in the human genome

To understand the architecture of the human genome, we need a complete definition of all the repeat sequence families, as these make up the majority of human DNA. We have isolated a small DNA fragment from human chromosome 21 and have used sequence analysis of this fragment to uncover a new low copy repeat element of approximately 300 bp that we term the Mermaid repeat. This repeat is related to, but is different from, the MER 12 repeat and is interspersed in the genome. Mermaid family members that we have studied are between 81%–87% identical to our preliminary consensus sequence. Therefore, we have added a new member to the large collection of human repetitive elements. In addition, we have mapped a Mermaid repeat to a telomeric position on the long arm of human chromosome 21, at 21q22.3     

Restriction enzyme analysis of satellite DNA components from the bovine genome

A restriction enzyme analysis was performed on satellite DNA components, isolated, as described in the preceding paper, from the bovine genome by a combination of Cs2SO4/BAMD and Cs2SO4/Ag+ density gradient centrifugation. Such an analysis has led to the unambiguous identification of eight satellite DNA components and to new information on their repeat units; this indicates that identical repeat lengths are shared by them, a fact strongly suggesting a common origin.     

Effects of sequence on repeat expansion during DNA replication

Small DNA repeat tracts are located throughout the human genome. The tracts are unstable, and expansions of certain repeat sequences cause neuromuscular disease. DNA expansions appear to be associated with lagging-strand DNA synthesis and DNA repair. At some sites of repeat expansion, e.g. the myotonic dystrophy type 2 (DM2) tetranucleotide repeat expansion site, more than one repeat tract with similar sequences lie side by side. Only one of the DM2 repeat tracts, however, is found to expand. Thus, DNA base sequence is a possible factor in repeat tract expansion. Here we determined the expansion potential, during DNA replication by human DNA polymerase β, of several tetranucleotide repeat tracts in which the repeat units varied by one or more bases. The results show that subtle changes, such as switching T for C in a tetranucleotide repeat, can have dramatic consequences on the ability of the nascent-strand repeat tract to expand during DNA replication. We also determined the relative stabilities of self-annealed 100mer repeats by melting-curve analysis. The relative stabilities did not correlate with the relative potentials of the analogous repeats for expansion during DNA replication, suggesting that hairpin formation is not required for expansion during DNA replication.     

Acquisition of telomere repeat sequences by transfected DNA integrated at the site of a chromosome break.

Previous analysis of plasmid DNA transfected into 108 cell clones demonstrated extensive polymorphism near the integration site in one clone. This polymorphism was apparent by Southern blot analysis as diffuse bands that extended over 30 kb. In the present study, nucleotide sequence analysis of cloned DNA from the integration site revealed telomere repeat sequences at the ends of the integrated plasmid DNA. The telomere repeat sequences at one end were located at the junction between the plasmid and cell DNA. The telomere repeat sequences at the other end were located in the opposite orientation in the polymorphic region and were shown by digestion with BAL 31 to be at the end of the chromosome. Telomere repeat sequences were not found at this location in the plasmid or parent cell DNA. Although the repeat sequences may have been acquired by recombination, a more likely explanation is that they were added to the ends of the plasmid by telomerase before integration. Comparison of the cell DNA before and after integration revealed that a chromosome break had occurred at the integration site, which was shown by fluorescent in situ hybridization to be located near the telomere of chromosome 13. These results demonstrate that chromosome breakage and rearrangement can result in interstitial telomere repeat sequences within the human genome. These sequences could promote genomic instability, because short repeat sequences can be recombinational hotspots. The results also show that DNA rearrangements involving telomere repeat sequences can be associated with chromosome breaks. The introduction of telomere repeat sequences at spontaneous or ionizing radiation-induced DNA strand breaks may therefore also be a mechanism of chromosome fragmentation.     

Telomere repeat DNA forms a large non-covalent complex with unique cohesive properties which is dissociated by Werner syndrome DNA helicase in the presence of replication protein A

We describe the unique structural features of a large telomere repeat DNA complex (TRDC) of >20 kb generated by a simple PCR using (TTAGGG)₄ and (CCCTAA)₄ as both primers and templates. Although large, as determined by conventional agarose gel electrophoresis, the TRDC was found to consist of short single-stranded DNA telomere repeat units of between several hundred and 3000 bases, indicating that it is a non-covalent complex comprising short cohesive telomere repeat units. S1 nuclease digestion showed that the TRDC contains both single- and double-stranded portions stable enough to survive glycerol density gradient centrifugation, precipitation with ethanol and gel electrophoresis. Sedimentation analysis suggests that a part of the TRDC is non-linear and consists of a three-dimensional network structure. After treatment with Werner DNA helicase the TRDC dissociated into smaller fragments, provided that human replication protein A was present, indicating that: (i) the TRDC is a new substrate for the Werner syndrome helicase; (ii) the telomere repeat sequence re-anneals rapidly unless unwound single-stranded regions are protected by replication protein A; (iii) the TRDC may provide a new clue to understanding deleterious telomere–totelomere interactions that can lead to genomic instability. Some properties of the TRDC account for the extra-chromosomal telomere repeat (ECTR) DNA that exists in telomerase-negative immortalized cell lines and may be involved in maintaining telomeres.     

Real-time detection of DNA topological changes with a fluorescently labeled cruciform

Topoisomerases are essential cellular enzymes that maintain the appropriate topological status of DNA and are the targets of several antibiotic and chemotherapeutic agents. High-throughput (HT) analysis is desirable to identify new topoisomerase inhibitors, but standard in vitro assays for DNA topology, such as gel electrophoresis, are time-consuming and are not amenable to HT analysis. We have exploited the observation that closed-circular DNA containing an inverted repeat can release the free energy stored in negatively supercoiled DNA by extruding the repeat as a cruciform. We inserted an inverted repeat containing a fluorophore-quencher pair into a plasmid to enable real-time monitoring of plasmid supercoiling by a bacterial topoisomerase, Escherichia coli gyrase. This substrate produces a fluorescent signal caused by the extrusion of the cruciform and separation of the labels as gyrase progressively underwinds the DNA. Subsequent relaxation by a eukaryotic topoisomerase, human topo IIα, causes reintegration of the cruciform and quenching of fluorescence. We used this approach to develop a HT screen for inhibitors of gyrase supercoiling. This work demonstrates that fluorescently labeled cruciforms are useful as general real-time indicators of changes in DNA topology that can be used to monitor the activity of DNA-dependent motor proteins.     

Mer22-related sequence elements form pericentric repetitive DNA families in primates

We describe a novel repetitive DNA element isolated from three primate species belonging to the family Cercopithecidae. The unusually long 2.6-kb repeat unit of this DNA element is present in high copy number in the pericentromeric region of one pair of chromosomes in both baboon and macaque, forming chromosome-specific satellite-like DNA families. Besides these two very closely related species, the novel DNA element was also detected in the more distantly related African green monkey. However, the copy number of the repeat unit in this species is significantly lower than in macaque and baboon. Sequence analysis revealed that the repeat units of the new repetitive element show similarity to the human MER22 repeat and the Y chromosome-specific TTY2 element, which also exhibits retroelement-like features. Database searches indicate that tandemly arranged MER22-related DNA sequences can also be found in human, raising the possibility that these DNA elements may correspond to a novel primate-specific repetitive DNA group. Recent studies indicate that chromosome-specific pericentric repetitive elements, besides their potential involvement in centromere function, also facilitate homolog recognition during meiosis. In addition, rapid expansion of retroelements in the pericentric regions of chromosomes during interspecific hybridization has been described. In light of these data, we hypothesize that the novel repetitive element described here might have been involved in the speciation of the family Cercopithecidae.     

Images of Alu-sequence in 7 DNA clones from the human genome

Information theory methods were used for computer search of Alu-like sequences in human DNA and RNA. Eight new regions related to the Alu repeat sequence was revealed in 85 clones from the EMBL-5 data bank. Some of these regions are purine-pyrimidine images of Alu repeats sequence, the rest are more complex images of Alu repeat sequence. A new definition for the likeness of different sequences--information image of sequence--was introduced. This information theory application greatly increases the power of DNA sequences computer analysis.     

Characterization and genetic mapping of a short, highly repeated, interspersed DNA sequence from rice (Oryza sativa L.).

Summary A short, highly repeated, interspersed DNA sequence from rice was characterized using a combination of techniques and genetically mapped to rice chromosomes by restriction fragment length polymorphism (RFLP) analysis. A consensus sequence (GGC)_n, where n varies from 13–16, for the repeated sequence family was deduced from sequence analysis. Southern blot analysis, restriction mapping of repeat element-containing genomic clones, and DNA sequence analysis indicated that the repeated sequence is interspersed in the rice genome, and is heterogeneous and divergent. About 200000 copies are present in the rice genome. Single copy sequences flanking the repeat element were used as RFLP markers to map individual repeat elements. Eleven such repeat elements were mapped to seven different chromosomes. The strategy for characterization of highly dispersed repeated DNA and its uses in genetic mapping, DNA fingerprinting, and evolutionary studies are discussed.     

A new source of polymorphic DNA markers for sperm typing: analysis of microsatellite repeats in single cells.

We show that dinucleotide and tetranucleotide repeat polymorphisms can be analyzed in single cells without using radioactivity or denaturing gels. This provides a rich new source of DNA polymorphisms for genetic mapping by sperm typing. The recombination fraction between two CA repeat polymorphisms was determined after whole genome amplification of single sperm, followed by typing of two different aliquots, one aliquot for each polymorphic locus. Single-cell analysis of microsatellites may also be valuable both for preimplantation genetic disease diagnosis based on single-blastomere or polar-body analysis and for the typing of forensic or ancient DNA samples containing very small amounts of nucleic acid.