卫星,小卫星和微卫星DNA—真核生物基因组的串状重复序列

卫星、小卫星和微卫星ＤＮＡ——真核生物基因组的串状重复序列姜运良（山东农业大学动物科技学院,山东泰安２７１０１８）关键词卫星小卫星微卫星串状重复序列真核生物基因组中编码蛋白质（酶）的结构基因只占很少的一部分（１０％～２０％）,其余大部分是重复序列。根...     

中国明对虾基因组小卫星重复序列分析

通过对中国明对虾基因组随机DNA片断的测序 ,我们获得了总长度约 6 4 10 0 0个碱基的基因组DNA序列 ,从中共找到 172 0个重复序列。其中 ,小卫星序列的数目为 398个 ,占重复序列总数目的 2 3 14 %。这些小卫星序列的重复单位长度为 7- 16 5个碱基 ,集中分布于 7- 2 1个碱基范围内 ,其中以重复单位长度为 12个碱基的重复序列数目最多 ,为 5 8个 ,占小卫星重复序列总数目的 14 5 7%。不同拷贝数目所对应的重复序列的数目情况为 :拷贝数目为 2的重复单位所组成的重复序列数目最多 ,为 137个 ;其次是拷贝数目为 3的重复序列 ,为12 2个 ,且随着拷贝数目的增加 ,由其所组成的重复序列的数目呈递减的趋势。其中一部分序列见GeneBank数据库 ,登录号为AY6 990 72 -AY6 990 76。 398个重复序列分别由 398种重复单位所组成 ,因而小卫星重复序列的类型很多 ,我们初步分成三类 :两种碱基组成类别、三种碱基组成类别和四种碱基组成类别 ,并进一步根据各个重复序列中所含有的碱基种类的数量从大到小排列这些碱基而分成若干小类。从这些分类中可以看出 ,中国明对虾基因组中的小卫星整体上是富含A T的重复序列 ,并具有一定的“等级制度” ,揭示了其与微卫星重复序列之间的关系 ,即一部分小卫星重复序列可能起源于微卫星     

蓝狐-卫星DNA的克隆及序列分析

利用限制性内切酶酶切蓝狐基因组, 经琼脂糖凝胶电泳, 对特异性亮带进行克隆、测序及序列分析。结果获得42个卫星DNA序列, 该卫星DNA单体大小为737 bp, G+C含量为51.9%, 单体之间同源性为91%~97%; 每个单体由3个约245 bp的亚重复串联构成, 亚重复之间的同源性为49%~55%; 在物种进化过程中, 该卫星DNA有G+C含量逐渐降低而A+T含量逐渐上升的趋势; 该卫星DNA为犬科动物种属所特有, 与犬着丝粒相关卫星DNA为同类卫星DNA, 同源性为74%, 命名为α-卫星DNA。     

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。     

纵纹腹小鸮DNA指纹谱探针的筛选和初步应用

以人工合成的微卫星序列 (GTG) 5,(GT) 8,(CAC) 5和人源小卫星 33 1 5作引物 ,扩增纵纹腹小的基因组DNA ,产生多态性DNA片段 ,回收了 8个表现个体特异性的片段。当用小的基因组总DNA探针与它们杂交时 ,其中 2个表现阳性 ,说明PCR方法扩增出的高变异产物含有重复序列。用含重复序列的个体特异性PCR产物作探针 ,与无关个体小基因组DNA的HaeⅢ酶切产物进行DNA印迹 ,获得了变异性较高的DNA指纹图谱。且通过对京白鸡家系分析表明 ,用小基因组DNA的PCR产物分离制备的探针所获得的DNA指纹图带能够稳定的遗传。因此 ,高变异的PCR产物可以有效地用作DNA指纹探针。     

鳄龟科和平胸龟科线粒体控制区序列分析和结构比较

本文参照龟类近缘种的线粒体DNA(mitochondrial DNA,mtDNA)控制区(control region,CR)及邻接序列,设计了二对特异引物,采用PCR和测序技术,获得了大鳄龟(Macroclemys temminckii)、小鳄龟(Chelydra serpentina)和平胸龟(Platysternon megacephalum)mtDNA CR区序列,其长度分别为1062bp、1124bp和1119bp;A T的含量分别为68.93%、69.34%和69.44%。序列分析显示,三种龟CR区3'末端均存在丰富的微卫星序列,其中大鳄龟和小鳄龟各有一段2bp的TA序列分别重复20和15次;小鳄龟另有一段5bp的TATAT序列重复13次;平胸龟则是一段10bp的AGTATGTTAT序列重复4次和一段17bp的GTTGTTATATAACATAT序列重复13次。本文还结合GenBank中已发表的其他6种龟鳖类动物的控制区序列,探讨了龟鳖类动物微卫星序列的类型及分布,结果表明:9种龟鳖类动物都存在丰富的微卫星序列,且微卫星所在位置及序列存在很大差异。     

纵纹腹小HaoDNA指纹谱探针的筛选和初步应用

以人工合成的微卫星序列（GTG）5,（GT）8,（CAC）5和人源小卫星33.15作引物,扩增纵纹腹小Hao的基因组DNA,产生多态性DNA片段,回收了8个表现个体特异性的片段,当用小Hao的基因组总DNA探针与它们杂交时,其中2个表现阳性,说明PCR方法扩增出的高变异产物含有重复序列,用含重复序列的个体特异性PCR产物作探针,与无关个体小Hao基因组DNA的HaeⅢ酶切产物进行DNA印迹,获得了变性性较高的DNA指纹图谱,且通过对京白鸡家系分析表明,用小Hao基因组DNA 的PCR产物分离制备的探针所获得的DNA指纹图带能够稳定的遗传,因此,高变异的PCR产物可以有效地用作DNA指纹探针。     

商城肥鲵二碱基重复和四碱基重复微卫星DNA的结构特征及对筛选效率的影响

两栖类有尾目物种的微卫星分离中的筛选成功率常常较低。为探索微卫星结构对筛选效率的影响,本研究通过AFLP快速分离法(fast isolation by AFLP of sequences containing repeats,FIASCO)对商城肥鲵(Pachyhynobius shangchengensis)二碱基重复类型和四碱基重复类型微卫星进行分离,并对微卫星序列进行了分析。研究中发现二碱基微卫星位点多以微卫星DNA家族形式存在,并因此导致了微卫星位点分离较低的筛选率;在四碱基重复的微卫星位点中未发现微卫星DNA家族的存在。对研究中得到的3个微卫星DNA家族的分析发现,同一家族的上、下游侧翼序列变异程度存在差异;毗邻微卫星重复单元区的侧翼序列碱基变异程度较高,而较远处的区段则相对保守。这些结构特征可能反映出微卫星DNA家族在演化中的复杂性。本文的研究结果提示在两栖动物的一些类群中,微卫星的筛选必须考虑微卫星DNA家族的影响,选取适宜的碱基重复类型将是决定筛选效率的关键。     

秦岭羚牛高度重复序列DNA片段的分子克隆和序列分析

羚牛(Budorcas taxicolor)属偶蹄目(Artiodactyla)、牛科(Bovidae),为我国一类大型珍贵保护动物。我们从其基因组中克隆得到若干约800bp的BamHI高度重复序列并对部分克隆进行了序列测定,发现它们显示了很高的同源性。利用其中一个单元为探针,对限制酶消化后的羚牛基因组DNA作杂交分析,发现其杂交谱带不具有个体及亚种间特异性,说明该重复序列在羚牛基因组中具有保守的分布和排列。在牛科动物中,羚牛BamHI片段与绵羊属和山羊属的相关序列具有高度同源性,而与水牛和家牛序列差异较大。这些结果为羚牛与羊亚科物种亲源关系较近的分类学观点提供了分子生物学证据。有证据表明,这些片段可能代表羚牛染色体着丝点的卫星DNA单体。     

巴氏蘑菇微卫星序列的分离及其对Co60辐射诱变菌株的鉴别

根据链霉素磁珠和生物素特异结合的特性,用生物素标记的二聚核苷酸重复序列探针从巴氏蘑菇的基因组中分离微卫星序列。将结合于链霉素磁珠上的标记探针同两端连接已知序列人工接头的巴氏蘑菇DNA酶切片段杂交。洗脱未杂交DNA片段后,用磁珠富集的片段建立微卫星文库。挑取522个菌落用对应重复序列为引物进行PCR筛选,得到48个阳性克隆,经测序有32个菌落含微卫星序列。微卫星富集效率为阳性克隆数的67%,总克隆数的6%。除去重复或无效的微卫星序列,在设计出的12对用于鉴别85个巴氏蘑菇的Co60辐射变异株微卫星引物中,有4对引物总共扩增出明显的变异菌株17个。证明有些微卫星位点可用于巴氏蘑菇辐射变异品种的指纹筛选与鉴别。     

Positive correlation between DNA polymerase alpha-primase pausing and mutagenesis within polypyrimidine/polypurine microsatellite sequences

Microsatellite DNA sequences are ubiquitous in the human genome, and mutation rates of these repetitive sequences vary with respect to DNA sequence as well as length. We have analyzed polymerase-DNA interactions as a function of microsatellite sequence, using polypyrimidine/polypurine di- and tetranucleotide alleles representative of those found in the human genome. Using an in vitro primer extension assay and the mammalian DNA polymerase alpha-primase complex, we have observed a polymerase termination profile for each microsatellite that is unique to that allele. Interestingly, a periodic termination profile with an interval size (9-11 nucleotides) unrelated to microsatellite unit length was observed for the [TC](20) and [TTCC](9) templates. In contrast, a unit-punctuated polymerase termination profile was found for the longer polypurine templates. We detected strong polymerase pauses within the [TC](20) allele at low reaction pH which were eliminated by the addition of deaza-dGTP, consistent with these specific pauses being a consequence of triplex DNA formation during DNA synthesis. Quantitatively, a strand bias was observed in the primer extension assay, in that polymerase synthesis termination is more intense when the polypurine sequence serves as the template, relative to its complementary polypyrimidine sequence. The HSV-tk forward mutation assay was utilized to determine the corresponding polymerase alpha-primase error frequencies and specificities at the microsatellite alleles. A higher microsatellite polymerase error frequency (50x10(-4) to 60x10(-4)) was measured when polypurine sequences serve as templates for DNA synthesis, relative to the polypyrimidine template (18x10(-4)). Thus, a positive correlation exists between polymerase alpha-primase pausing and mutagenesis within microsatellite DNA alleles.     

Silene tatarica microsatellites are frequently located in repetitive DNA

The genomic distribution of microsatellites can be explained by DNA slippage, slippage like processes and base substitutions. Nevertheless, microsatellites are also frequently associated with repetitive DNA, raising the question of the relative contributions of these processes to microsatellite genesis. We show that in Silene tatarica about 50% of the microsatellites isolated by an enrichment cloning protocol are associated with repetitive DNA. Based on the flanking sequences, we distinguished seven different classes of repetitive DNA. PCR primers designed for the flanking sequences of an individual clone amplified a heterogeneous family of repetitive DNA. Despite considerable variation in the flanking sequence (pi = 0.108), the microsatellite repeats did not show any evidence for decay. Rather, we observed the emergence of a new repeat type that probably arose by mutation and was spread by replication slippage. In fact, a complete repeat type switch could be observed among the analysed clones. We propose that the analysis of microsatellite sequences embedded in repetitive DNA provides a hitherto largely unexplored tool to study microsatellite evolution.     

Characterization of swine short interspersed repetitive sequences

Swine genomic DNA segments containing repetitive sequences were isolated from a porcine genomic library using genomic DNA as a probe. Three fragments containing the repetitive sequences from two of the primary phage clones were subcloned for sequence analysis, which revealed six new PRE-1 repetitive families other than those reported earlier by Singer et al. (Nucleic Acids Research 15, 2780, 1987). The frequency of the repetitive sequences in the swine genome was estimated at 2 x 10(6) per diploid genome. Sequence analysis revealed similarities between these repetitive sequences and that of arginine-tRNA gene.     

Repetitive DNA of grapevine: classes present and sequences suitable for cultivar identification

Summary Repetitive DNA sequences present in the grapevine genome were investigated as probes for distinguishing species and cultivars. Microsatellite sequences, minisatellite sequences, tandemly arrayed genes and highly repetitive grapevine sequences were studied. The relative abundance of microsatellite and minisatellite DNA in the genome varied with the repeat sequence and determined their usefulness in detecting RFLPs. Cloned Vitis ribosomal repeat units were characterised and showed length heterogeneity (9.14–12.15 kb) between and within species. A highly repetitive DNA sequence isolated from V. vinifera was found to be specific only to those species classified as Euvitis. DNA polymorphisms were found between Vitis species and between cultivars of V. vinifera with all classes of repeat DNA sequences studied. DNA sequences suitable for DNA fingerprinting gave genotype-specific patterns for all of the cultivars and species examined. The DNA polymorphisms detected indicates a moderate to high level of heterozygosity in grapevine cultivars.On leave from the Biochemical Research Institute, Nippon Menard Cosmetic Co, Ltd, Ogaki Gifuken, 503 Japan     

Microsatellite instability induced by hydrogen peroxide in Escherichia coli

Damage to DNA by reactive oxygen species may be a significant source of endogenous mutagenesis in aerobic organisms. Using a selective assay for microsatellite instability in E. coli, we have asked whether endogenous oxidative mutagenesis can contribute to genetic instability. Instability of repetitive sequences, both in intronic sequences and within coding regions, is a hallmark of genetic instability in human cancers. We demonstrate that exposure of E. coli to low levels of hydrogen peroxide increases the frequency of expansions and deletions within dinucleotide repetitive sequences. Sequencing of the repetitive sequences and flanking non-repetitive regions in mutant clones demonstrated the high specificity for alterations with the repeats. All of the 183 mutants sequenced displayed frameshift alterations within the microsatellite repeats, and no base substitutions or frameshift mutations occurred within the flanking non-repetitive sequences. We hypothesize that endogenous oxidative damage to DNA can increase the frequency of strand slippage intermediates occurring during DNA replication or repair synthesis, and contribute to genomic instability.     

Repetitive flanking sequences (ReFS): novel molecular markers from microsatellite families

Although microsatellite markers have become exceedingly popular in molecular studies of wild organisms, their development in some taxonomic groups is challenging. This is partly because of repetitive flanking sequences, which lead to the simultaneous amplification of alleles from multiple loci. Until now, these microsatellite DNA families have been considered unsuitable for population genetics studies, but here we describe our development of these repetitive flanking sequences (ReFS) as novel molecular markers. We illustrate the utility of these markers by using them to address an outstanding taxonomic question in the moth genus Schrankia.     

水稻BAC在玉米有丝分裂染色体上FISH杂交体系的构建

 以水稻细菌人工染色体(BAC)为探针在玉米有丝分裂的细胞学制片上进行荧光原位杂交(FISH),探讨玉米基因组C_ot DNA对BAC探针重复序列的封阻、杂交后洗脱的严谨度、杂交液中FAD的浓度变化、水稻BAC探针的特异性重复序列的封阻对FISH杂交信号特异性的影响.初步形成了一套以水稻BAC探针在玉米有丝分裂染色体上进行BAC-FISH杂交的优化技术体系.研究结果表明：使用玉米基因组C_ot DNA来封阻水稻BAC探针的重复序列玉米基因组C _ot DNA的C_ot值应小于50,同时还需根据不同探针调整C_ot DNA的C_ot值及与探针的比例;而降低杂交液中FAD浓度和适度控制杂交后洗脱的严谨度,尤其是使用水稻BAC探针本身特异的重复序列的封阻对BAC-FISH杂交信号特异性的改善具有较好的效果.     

鳞翅目昆虫基因组中微卫星DNA的特征以及对其分离的影响

本文根据我们对鳞翅目昆虫棉铃虫和松毛虫以及其它动物 (筏蜘蛛、朱、鳕鱼和飞蝗 )的微卫星富集性基因组DNA文库的筛选和分析结果 ,结合其它实验室已发表的资料 ,对鳞翅目昆虫基因组中微卫星DNA的丰度和结构特点进行了较为系统的分析。结果表明 :与其它类群相比 ,尽管鳞翅目昆虫物种间存在差异 ,但其基因组中存在明显偏多的侧翼序列重复的、以多拷贝形式存在的微卫星位点 ,且其中相当一部分以基因家族的形式存在。微卫星DNA家族通常可以在序列分析阶段被识别出来 ,但很多多拷贝位点只有通过一系列后续分析才能被检查出来。这应是鳞翅目昆虫中微卫星位点的优化率相对偏低的主要原因。棉铃虫和松毛虫基因组中三相重复微卫星丰度相对较高 ,从而从某种程度上补偿了这些物种微卫星分离过程中因丰度低、多拷贝位点比例高所带来的困难。棉铃虫微卫星DNA家族侧翼序列中多聚T/A序列的存在表明 ,逆转录转座或逆转录侵染可能是在基因组中形成多拷贝微卫星位点和微卫星DNA家族的重要机制之一     

DNA changes involving repeated sequences in senescing soybean (Glycine max) cotyledon nuclei

Age-related DNA changes in soybean ( Glycine max L. cv. Ransom) cotyledon nuclei were investigated by Feulgen cytophotometry and cloned DNA probes. The amount of nuclear DNA in 17-day-old senescing cotyledons was 23% lower than that of 5-day-old young cotyledons. In order to understand the nature of senescence-related DNA loss, fragments of repetitive DNA from young cotyledons were cloned into Escherichia coli HB101 cells by DNA recombination. The cloned DNA probed changes in specific repeated nucleotide sequences in senescing cotyledons. The colony hybridization between cloned DNA and [³²P]-labeled total DNA from 5- and 17-day cotyledons indicated loss of specific repeated sequences. The selected sequences of repeated DNA further showed a loss in their copy numbers. The study suggested that some repetitive DNA sequences were degraded selectively in the genome of senescing cotyledons.