高GC含量的鳜鱼rRNA基因家族的克隆

动物rRNA基因是一种GC含量较高、结构复杂的重复序列。该研究结合亲缘生物法生物信息学技术,经反复摸索后选用LA PCR法即LA PCR Taq酶结合GC缓冲液来扩增鳜鱼复杂的rRNA基因重复序列,经测序鉴定最终克隆了鳜鱼的3个rRNA基因及其2个间隔序列。分析了鳜鱼与相关动物的rRNA基因序列的同源性和进化关系。探索了克隆复杂DNA序列时引物设计的特别规则、反应体系的改进、DNA聚合酶的选用、循环参数的调整等措施。     

细菌基因组重复序列PCR技术及其应用

细菌中散在分布的DNA重复序列近年来不断被报道,基因外重复回文序列和肠细菌基因间共有重复序列是两个典型的原核细胞基因组散在重复序列。重复序列在染色体上的分布和拷贝数具种间特异性,用它们的互补序列作为引物,以细菌基因组DNA为模板进行PCR扩增反应,反应产物的琼脂糖电泳可以提供非常清晰的DNA指纹图谱,使用此图谱既可对各种微生物进行快速分型及鉴定,又可对它们进行DNA水平上的遗传多样性分析。细菌基因组重复序列PCR技术具有简捷、快速、结果稳定等特点,可对细菌进行分子标记,用于菌株分型、分类鉴定和亲缘关系等方面的研究。     

四核苷酸重复序列单链扩增特性及机理探究

简单重复序列广泛存在于多种生物基因组中,其生物学意义越来越受到人们的重视.许多简单重复序列易于扩增变长,某些重复序列的异常延伸是造成一些遗传疾病的直接原因.本研究以20 nt的60种四重复和6种二重复序列单链为模板,系统研究了它们在嗜热DNA聚合酶作用下等温扩增的特点.电泳结果显示,多数单链模板能扩增变长,即使链内没有互补碱基的序列也可被扩增,如(AGGA)5.定量分析结果显示:回文序列扩增最快;二重复序列比相同碱基组成的四重复序列有更宽的适于扩增的温度范围;G和C含量多的DNA较G和C含量少的序列更易扩增,而且G和C含量越多越适于在较高的温度下扩增;重复单位含两相同嘧啶的链多数比其互补链更易扩增;产物浓度与时间基本呈线性关系.限制性酶切产物结果显示,扩增产物与模板具有相同的重复单位,是重复序列的简单延伸.最后,根据实验结果和相关文献,提出了包括链内滑动扩增和发卡DNA介导扩增两阶段的重复序列单链扩增模型,以对重复序列非特异扩增和相关疾病发生机制的研究提供参考.     

高等植物DNA重复序列的主要类型和特点

高等植物核基因组的一个显著特征是其内含有大量的ＤＮＡ重复序列,因此它们在核基因组结构和功能研究中居于举足轻重的地位。一些ＤＮＡ重复序列已日趋广泛地作为分子民用于构建遗传图谱、鉴别品种、研究进化和分离目标基因等。主要介绍高等植物几类重要ＤＮＡ重复序列,如卫星ＤＮＡ、微卫星ＤＮＡ、核糖体ＲＮＡ基因、端粒重复序列和转座子等的若干特点和用途。     

3大种群蛋白质内部重复片段组成规律的比较研究

20世纪70年代,Ohno提出了功能蛋白的起源理论,认为寡肽片段的周期性重复是蛋白质起源的一种方式。蛋白质内部重复片段在蛋白质序列进化的过程中具有重要意义。选取原核生物、古细菌、真核生物的8个代表物种,设计了新的蛋白质内部重复片段的提取方法,并用矩阵的方式对重复片段的类型及其出现的频率进行形象地展现,既保留了重复片段的序列特征又可进行全局性的统计描述。分析表明：真核生物高频率的使用简单重复序列;真细菌也具有低频率使用简单重复序列的现象;而古细菌则几乎没有。进一步研究显示,3大种群生物偏向性使用氨基酸构成蛋白质内部重复片段的形为与蛋白质组的氨基酸使用频率紧密相关。其相关系数在真细菌和古细菌中高于0．95,而真核生物略低。真核生物蛋白质组大量使用简单重复片段,以及两者在氨基酸使用上的较低相关性暗示简单重复序列的快速进化是导致真核生物蛋白质组高复杂性的一个关键因素。     

人腺病毒B、D亚种基因组中简单重复序列分析

本文以人腺病毒B亚种31条基因组序列及D亚种39条基因组序列为研究材料,利用ImperfectMicrosatelliteExtractor和DNAMAN软件对这些基因组序列中简单重复序列(SSR)的分布情况进行了系统性分析和比较。分析结果显示:人腺病毒B、D亚种基因组中简单重复序列的平均相对密度是十分接近的,但在不同类型SSR中分布情况又有所不同。D亚种中二型SSR明显高于B亚种,在两亚种一型SSR中(A)n、(T)n都是比较多的,而在两亚种二型SSR中的(CG/GC)n表现出了较高的偏好性。在同亚种多序列比对分析中,D亚种表现出了更高的稳定性。B、D亚种中SSR的这种特异性分布可能与它们的进化机制和致病性有关。     

简单重复DNA序列在哺乳动物mtDNA D-loop区的分布及进化特征

简单重复序列广泛分布于从原核到真核生物的基因组中, 其形成的分子机理目前尚不明确。对NCBI数据库中已有256种哺乳动物线粒体DNA (mtDNA) D-loop区进行序列比对分析, 根据其所含有的简单重复序列类型分为3组, 分别是53种哺乳动物含有六核苷酸重复序列; 104种哺乳动物含有非六核苷酸重复序列(＞6 bp); 99种哺乳动物不含有任何重复序列。通过碱基序列分析比对, 发现六核苷酸重复序列集中分布在CSB1-CSB2间隔区, 而非六核苷酸重复可以分布于终止区(TAS)、中央保守区(Central domain)以及CSB(Central sequence block)区。通过比较含有重复序列与不含重复序列的功能保守区发现, 简单重复序列的存在并不明确影响D-loop区内的中央保守区以及CSB1、CSB2、CSB3三个功能保守区的碱基序列保守性。在此基础上, 利用N-J法构建了256种哺乳动物的进化树, 分析了哺乳动物D-Loop区内重复序列在进化过程中的可能变化规律, 发现简单重复序列随着物种的进化地位的升高而呈现消失趋势。     

一种基于二代测序辨识短串联重复序列长度变异的新方法及其在人类遗传疾病研究中的应用

二代测序技术的涌现推动了基因组学研究,特别是在疾病相关的遗传变异研究中发挥了重要作用．虽然大多数遗传变异类型都可以借助于各种二代测序分析工具进行检测,但是仍然存在局限性,比如短串联重复序列的长度变异．许多遗传疾病是由短串联重复序列的长度扩张导致的,尤其是亨廷顿病等多种神经系统疾病．然而,现在几乎没有工具能够利用二代测序检测长度大于测序读长的短串联重复序列变异．为了突破这一限制,我们开发了一个全新的方法,该方法基于双末端二代测序辨识短串联重复序列长度变异,并可估计其扩张长度,将其应用于一项基于全外显子组测序的运动神经元疾病临床研究中,成功地鉴定出致病的短串联重复序列长度扩张．该方法首次原创性地利用测序读长覆盖深度特征来解决短串联重复序列变异检测问题,在人类遗传疾病研究中具有广泛的应用价值,并且对于其他二代测序分析方法的开发具有启发性意义．     

串联重复序列在琴叶拟南芥基因组中的特征探究

串联重复序列广泛存在于真核生物的基因组中,它通过影响染色质的空间结构及基因表达从而影响生物的遗传与进化.本研究以琴叶拟南芥(Arabidopsis lyrata)基因组为材料,分析了1～50 bp重复单元的串联重复序列特征.研究发现串联重复序列在基因的5'UTR和启动子区域密度最高(8757 bp/Mb,8430 bp/Mb),而编码区CDS的密度最低(2406 bp/Mb).基因组中重复模体最高的为单核苷酸重复的T/A碱基,5'UTR中包含大量的二核苷酸重复模体,而在CDS中主要是三核酸重复模体.串联重复序列特征在琴叶拟南芥基因组不同区域的差别,显示其与基因表达和调控功能相适应.本研究深入探讨了串联重复序列在植物基因组中的特征及作用,为重复序列调控基因表达及植物基因组进化提供借鉴.     

基于转录组数据的荔枝蒂蛀虫SSR位点信息分析

荔枝蒂蛀虫Conopomorpha sinensis Bradley是专一性危害我国荔枝和龙眼的重要害虫。简单重复序列标记(Simple sequence repeat,SSR)为短串联重复序列或微卫星标记,其在荔枝蒂蛀虫偏嗜选择寄主的遗传进化机制研究和荔枝蒂蛀虫综合治理中具有重要意义。本研究基于高通量测序获得的荔枝蒂蛀虫转录组数据,利用MISA软件从68996条转录组unigenes结果中发掘出10521个SSR位点,出现频率为15.25%。荔枝蒂蛀虫转录组中SSR的主要重复类型为单碱基重复,占SSR总数的66.22%。其次是三碱基重复,占SSR总数的24.94%。在发现的33种重复基元中共筛选获得8种优势重复基元,其中A/T在单碱基重复基元中所占的比例达98.55%。基于筛选的SSR设计的9对引物中,有4对引物得到扩增预期大小的条带。荔枝蒂蛀虫SSR位点的信息分析将为探究荔枝蒂蛀虫种群遗传结构、遗传多样性和进化关系、害虫综合治理等研究提供重要科学依据。     

DNA ligase I competes with FEN1 to expand repetitive DNA sequences in vitro

Repeat sequences in various genomes undergo expansion by poorly understood mechanisms. By using an oligonucleotide system containing such repeats, we recapitulated the last steps in Okazaki fragment processing, which have been implicated in sequence expansion. A template containing either triplet or tandem repeats was annealed to a downstream primer containing complementary repeats at its 5'-end. Overlapping upstream primers, designed to strand-displace varying numbers of repeats in the downstream primer, were annealed. Human DNA ligase I joined overlapping segments of repeats generating an expansion product from the primer strands. Joining efficiency decreased with repeat length. Flap endonuclease 1 (FEN1) cleaved the displaced downstream strand and together with DNA ligase I produced non-expanded products. However, both expanded and non-expanded products formed irrespective of relative nuclease and ligase concentrations tested or enzyme addition order, suggesting the pre-existence and persistence of intermediates leading to both outcomes. FEN1 activity decreased with the length of repeat segment displaced presumably because the flap forms structures that inhibit cleavage. Increased MgCl(2) disfavored ligation of substrate intermediates that result in expansion products. Examination of expansion in vitro enables dissection of substrate and replication enzyme dynamics on repeat sequences.     

Elongation of repetitive DNA by DNA polymerase from a hyperthermophilic bacterium Thermus thermophilus

Short repetitive DNA sequences are believed to be one of the primordial genetic elements that served as a source of complex large DNA found in the genome of modern organisms. However, the mechanism of its expansion (increase in repeat number) during the course of evolution is unclear. We demonstrate that the DNA polymerase of the hyperthermophilic bacterium Thermus thermophilus can elongate oligoDNA with several tandem repeats to very long DNA in vitro. For instance, 48mer repetitive oligoDNA (TACATGTA)₆, which has 25% GC content and a palindromic sequence, can be elongated up to ~10 000 bases by DNA polymerase at 74°C without template DNA. OligoDNA having a different GC content or a quasi-palindromic sequence can also be elongated, but less efficiently. A spectroscopic thermal melting experiment with the oligoDNA showed that its hairpin–coil transition temperature was very close to the elongation reaction temperature (74°C), but was much higher than the temperature at which duplex oligoDNA can exist stably. Taken together, we conclude that repetitive oligoDNA with a palindromic or quasi-palindromic sequence is elongated extensively by a hyperthermophilic DNA polymerase through hairpin–coil transitions. We propose that such an elongation mechanism might have been a driving force to expand primordial short DNA.     

Characterization of a family of repetitive sequences that is eliminated late during macronuclear development of the hypotrichous ciliate Stylonychia lemnae

A repetitive element from the hypotrichous ciliate Stylonychia lemnae was characterized by restriction and hybridization analysis. This repetitive element is present in about 5,000–7,000 copies per haploid genome in the micronucleus and the macronuclear anlagen. Its DNA sequence is very conserved, but the length of the repetitive sequence blocs is variable. In some cases, it is associated with telomeric sequences and macronucleus–homologous sequences. Restriction analysis of genomic micronuclear and macronuclear anlagen DNA and in situ hybridization showed that the repetitive sequences are amplified during the formation of polytene chromosomes. They are localized in many bands of the polytene chromosomes and are eliminated during the degradation of the polytene chromosomes. Possible functions of the repetitive sequences during macronuclear differentiation are discussed. Dev. Genet. 21:201–211, 1997.© 1997 Wiley-Liss, Inc.     

Repetitive DNA and chromosome evolution in plants

Most higher plant genomes contain a high proportion of repeated sequences. Thus repetitive DNA is a major contributor to plant chromosome structure. The variation in total DNA content between species is due mostly to variation in repeated DNA content. Some repeats of the same family are arranged in tandem arrays, at the sites of heterochromatin. Examples from the Secale genus are described. Arrays of the same sequence are often present at many chromosomal sites. Heterochromatin often contains arrays of several unrelated sequences. The evolution of such arrays in populations is discussed. Other repeats are dispersed at many locations in the chromosomes. Many are likely to be or have evolved from transposable elements. The structures of some plant transposable elements, in particular the sequences of the terminal inverted repeats, are described. Some elements in soybean, antirrhinum and maize have the same inverted terminal repeat sequences. Other elements of maize and wheat share terminal homology with elements from yeast, Drosophila, man and mouse. The evolution of transposable elements in plant populations is discussed. The amplification, deletion and transposition of different repeated DNA sequences and the spread of the mutations in populations produces a turnover of repetitive DNA during evolution. This turnover process and the molecular mechanisms involved are discussed and shown to be responsible for divergence of chromosome structure between species. Turnover of repeated genes also occurs. The molecular processes affecting repeats imply that the older a repetitive DNA family the more likely it is to exist in different forms and in many locations within a species. Examples to support this hypothesis are provided from the Secale genus.     

Nucleotide sequence analysis of a mouse Y chromosomal DNA fragment containing Bkm and LINE elements

The strong suppression of crossing-over between the X and Y chromosomes permits rapid accumulation of repetitive sequences in the Y chromosome. To gain insight into the mechanism responsible for the sequence amplification, it is essential to characterize Y chromosomal repetitive sequences at the molecular level. Here, we report the entire nucleotide sequence (3,902bp) of AC11, a mouse sequence that is repeated 300 times in the Y chromosome. AC11 is AT rich (32.8% GC), and contains many short poly(A) sequences. In addition, it has Bkm and LINE sequences as well as a Y chromosome-specific sequence. The Bkm sequence consists of typical (GATA) and (GACA) repeating units, whereas the LINE sequence deviates considerably from other mouse LINE sequences (71–76% identity) and may be considered atypical. The Y chromosome-specific region seems to be unique and does not identify similar sequences in the GenBank library. The information obtained from the nucleotide sequence should form the foundation to study the evolutionary processes through which AC11-related sequences have accumulated in the mouse Y chromosome.     

The ligation amplification reaction (LAR)--amplification of specific DNA sequences using sequential rounds of template-dependent ligation

A novel DNA sequence detection method that utilizes the ligation of oligonucleotide pairs that are complementary to adjacent sites on appropriate DNA templates is described. The product is increased by either linear or exponential amplification using sequential rounds of template-dependent ligation. In the case of linear amplification, a single pair of oligonucleotides is ligated, the reaction is heated to dissociate the ligation product, and an additional round of ligation is performed. After n rounds there is a (1 + x) X n-fold amplification of product, where x is the efficiency of the ligation reaction. Exponential amplification utilizes two pairs of oligonucleotides, one complementary to the upper strand and one to the lower strand of a target sequence. The products of the ligation reaction serve as templates for subsequent rounds of ligation. In this case there is (1 + x)(n-1)-fold amplification of product after n rounds. A single base-pair mismatch between the annealed oligonucleotides and the template prevents ligation, thus allowing the distinction of single base-pair differences between DNA templates. At high template concentrations, the ligation reaction has an efficiency approaching 100%. In this report, we demonstrate the use of the ligation amplification reaction (LAR) to distinguish the normal from the sickle cell allele of the human beta-globin gene. We also report the use of LAR as a detection system for polymerase chain reaction-enriched DNA sequences.     

PlantSat: a specialized database for plant satellite repeats

MOTIVATION: Tandemly organized repetitive sequences (satellite DNA) are widespread in complex eukaryotic genomes. In plants, satellite repeats often represent a substantial part of nuclear DNA but only a little is known about the molecular mechanisms of their amplification and their possible role(s) in genome evolution and function. Unfortunately, addressing these questions via characterization of general sequence properties of known satellite repeats has been hindered by a difficulty in obtaining a complete and unbiased set of sequence data for this analysis. This is mainly due to the presence of multiple entries of homologous sequences and of single entries that contain more than one repeated unit (monomer) in the public databases. RESULTS: We have established a computer database specialized for plant satellite repeats (PlantSat) that integrates sequence data available from various resources with supplementary information including repeat consensus sequences, abundances, and chromosomal localizations. The sequences are stored as individual repeat monomers grouped into families, which simplifies their computer analysis and makes it more accurate. Using this feature, we have performed a basic sequence analysis of the whole set of plant satellite repeats with respect to their monomer length and nucleotide composition. The analysis revealed several preferred length ranges of the monomers (approximately 165 bp and its multiples) and an over-representation of the AA/TT dinucleotide in the repeats. We have also detected an enrichment of satellite DNA sequences for the motif CAAAA that is supposed to be involved in breakage-reunion of repeated sequences.     

Characteristics and a comparison of three classes of microsatellite-based markers and their application in plants

Microsatellites (SSR--simple sequence repeats, STR--short tandem repeats, SSLP--simple sequence length polymorphism, VNTR--variable number of tandem repeats) are the class of repetitive DNA sequences present in all living organisms. Particular characteristics of microsatellites, such as their presence in the genomes of all living organisms, high level of allelic variation, co-dominant mode of inheritance and potential for automated analysis make them an excellent tool for a number of approaches like genotyping, mapping and positional cloning of genes. The three most popular types of markers containing microsatellite sequences that are presently used are: (1) SSR (simple sequence repeats), generated by amplifying in a PCR reaction with the use of primers complementary to flanking regions; (2) ISSR (inter-simple sequence repeats), based on the amplification of regions between inversely oriented closely spaced microsatellites; and (3) SAMPL (selective amplification of microsatellite polymorphic loci), which utilises AFLP (amplified fragment-length polymorphism) methodology, with one exception--for the second amplification, one of the starters is complementary to the microsatellite sequence. The usefulness of the three above-mentioned markers for numerous purposes has been well documented for plants.     

Cytological characterization of the tandem repetitive sequences and their methylation status in the Antirrhinum majus genome

Tandem repetitive sequences are DNA motifs common in the genomes of eukaryotic species and are often embedded in heterochromatic regions. In most eukaryotes, ribosomal genes, as well as centromeres and telomeres or subtelomeres, are associated with abundant tandem arrays of repetitive sequences and typically represent the final barriers to completion of whole-genome sequencing. The nature of these repeats makes it difficult to estimate their actual sizes. In this study, combining the two cytological techniques DNA fiber-FISH and pachytene chromosome FISH allowed us to characterize the tandem repeats distributed genome wide in Antirrhinum majus and identify four types of tandem repeats, 45S rDNA, 5S rDNA, CentA1, and CentA2, representing the major tandem repetitive components, which were estimated to have a total length of 18.50 Mb and account for 3.59% of the A. majus genome. FISH examination revealed that all the tandem repeats correspond to heterochromatic knobs along the pachytene chromosomes. Moreover, the methylation status of the tandem repeats was investigated in both somatic cells and pollen mother cells from anther tissues using an antibody against 5-methylcytosine combined with sequential FISH analyses. Our results showed that these repeats were hypomethylated in anther tissues, especially in the pollen mother cells at pachytene stage.