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。     

用寡核苷酸探针（CAC）5／（GTG）5进行人的DNA指纹分析

在人或动物的基因组中,存在着类似于小卫星DNA的简单衔接重复单位,如(GACA)_4、(GATA)_4、(TCC)_5、(CA)_8和(CAC)_5等,由于这些重复单位在人或动物基因组中出现的数目和频率不同而表现出多态性。本文用人工合成的寡核苷酸探针(CAC)_5/(GTG)_5,调查了北京地区的50名无关个体,经过统计学处理,计算出无关个体的相关机率是3.8×10~(-10);此外,还对2个家系中的11名成员和1对双胞胎进行了检测,其结果显示子代中的杂交带分别来自父亲和母亲;双胞胎的DNA指纹图完全一致。研究结果表明,(CAC)_5/(GTG)_5探针检出的谱带具有高度的个体特异性(同卵双生子除外),并且谱带在亲代与子代间的传递符合孟德尔遗传规律。     

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。     

用随机扩增多态性DNA产物做探针产生鸡的DNA指纹图

我们用12个随机扩增多态性DNA(RAPD)引物对来自不同品系的4只鸡进行了RAPD分析,在扩增出的共99条带中,表现多态性的带为38条,占总带数的38％.回收了4个表现个体特异性的RAPD产物,当用鸡的基因组总DNA探针与它们杂交时,其中3个表现阳性,说明RAPD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RAPD产物作探针,与无关个体鸡基因组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.因此,高变异的RAPD产物可以有效地用作DNA指纹探针.     

中国稻瘟病菌的致病性和其DNA指纹图谱间关系的研究

通过在稻瘟病菌Pyricularia oryza。基因组文库中的筛选,找到一个散布的并具有基因组特异性的层重复顺序POR6。本文报道用这一株针对6个日本菌株和26个中国北方菌株进行DNA指纹作图的结果。其中22个中国北方菌株按其杂交带型百分相似率被分成8个株系。一些在我们实验室保存的菌株用传统方法鉴定发现在转管过程中会发生致病性变异。当用POR6作探针与这些菌株NNA的EcoRV酶切片段杂交时,检测出它们的无性世代中出现数条EcoRV多态性片段。     

人类染色体8q24.1带特异性微小卫星DNA的筛选

本研究运用人类高分辨染色体显微切割、ＰＣＲ技术获得的８ｑ２４．１带特异性探针池,构建了该区带的ｐＵＣ１９文库,从中筛选出４８个含ＣＡ重复顺序的微小卫星ＤＮＡ的克隆,已完成１２个克隆的序列分析,发现了一个世界上至今未曾报道过的、在正常人群中已检出１１个等位片段的、杂合度在中国汉族人群和美国盎格鲁撤克逊族人群中分别达０．８４和０．８３的高度多态的微小卫星ＤＮＡ（编号：Ｄ８Ｓ７Ｆ）,经ＰＣＲ检测人鼠杂种细胞系列,证实其来源于人８号染色体。     

寡聚核苷酸探针在近交系小鼠遗传检测中的应用

用两个非放射性标记的寡聚核苷酸探针(GGAT)4和(GTG)5制作BALB/c、C57BL/6J、DBA/2、C3H等4种近交系小鼠的DNA指纹图,比较了两种探针在近交系小鼠遗传检测应用中的重复性和稳定性。结果表明,两种探针对上述4种近交系小鼠产生的DNA指纹图的图带数均为8～12条,具有良好的多态性。品系内的平均DNA指纹图相似系数(-x)在0·92～1·00的范围内,具有相同指纹图的概率(P)均在0·31以上,极显著地高于品系间的相似系数(0·22～0·39)和相同指纹图的概率(P<1·07×10-4)。说明(GGAT)4和(GTG)5两种寡聚核苷酸探针均可用于制作近交系小鼠的DNA指纹图,以对其进行遗传检测。用两种不同的探针进行DNA指纹分析,可以检出基因组中更多的个体特异性信息,结果更加可靠。     

恒河猴群微卫星DNA多态性的分析

目的　确立一种对恒河猴群个体的遗传物质进行准确可靠、快速简便的遗传检测方法。方法　利用聚合酶链反应 (PCR)扩增技术对 2 0只恒河猴群个体间进行了DNA多态性的分析。结果　筛选出 9个微卫星DNA位点具有显著多态性 ,4个微卫星DNA位点没有多态性 ,还有 2个位点等位基因数目较少。结论　利用这些多态性微卫星位点建立一种对恒河猴群个体进行有效、准备可靠、快捷简便的遗传背景监测方法。     

Y染色体特异DNA序列及应用

DNA文库的建立及其应用于基因定位的可能性首先在基因组较小的生物(如果蝇)中得到证实。近年来原位噬菌斑和克隆杂交,改进的λ克隆载体以及体外包装系统等的发展,对于较复杂的基因组(哺乳动物)也进行了基因文库的建立和筛选。对复杂基因组中富集某一特定部分的文库的建立开始于七十年代末,被克隆的特定染色体DNA顺序文库代表全部基因组信息的结构亚单位,比整个基因组定位具有显著的优点。     

一些水稻的重复DNA顺序在稻属和其它禾本科植物中的多态性

重复DNA顺序是真核生物基因组的特征,很多重复DNA顺序已从小麦、拟南芥菜、燕麦、水稻、玉米等植物的基因组中克隆出来,还发现有一些重复DNA顺序具有基因组特异性,用它们作探针可以分析同属或同科物种的起源和亲缘关系,并建立系统进化树。小卫星DNA或微小卫星DNA所产生的指纹图谱可作为一种遗传学标志来研究系统进化、染色体的精细结构和物种的鉴定。一些中度重复DNA序列还可以作为组织培养株系和细胞杂交筛选的分子标志。稻属已发现并定名的有22个种,根据杂交亲和性、细胞遗传学和生理生化等将它分为6个二倍体组型(AA、BB、CC、DD、EE和FF)和2个四倍体组型(BBCC和CCDD)。现多把禾本科分作5个亚科:竹亚科、稻亚科、早熟禾亚科、画眉草亚科和     

Identification of new minisatellites loci in Arabidopsis thaliana

In order to characterize new CG-rich minisatellites present in the Arabidopsis thaliana genome, a genomic library was screened at low stringency with a probe containing nine repeated-units of a minisatellite (CMs1) previously identified. Both minisatellites and minisatellite-like elements were identified. The minisatellites, with a tandemly-repeated structure, all contain the Arabidopsis thaliana-core sequence previously defined (Tourmente et al., 1994). Both minisatellite and minisatellite-like sequences occur in the Arabidopsis genome in low copy and are weakly polymorphic between ecotypes. The genetic mapping of these markers has shown that they are dispersed on the genome. YACs clones of the CIC library carrying these minisatellites and minisatellite-like sequences were identified.Key words: Arabidopsis thaliana, minisatellites, polymorphism      

Identification, isolation and characterization of canine minisatellite sequences

A Charomid ordered-array library containing a 2–16 Kb size fraction of MbeoI-digested canine genomic DNA has been screened with the Jeffreys multilocus probes, 33-6 and 33-15, to identify and isolate canine minisatellite sequences. Of the 48 positive clones identified, 7 were found to contain polymorphic mini-satellites with heterozygosities in the range 20–88%. The majority of the remainder were either monomorphic or dimorphic in the animals tested. Analysis of intrabreed variation in Bedlington Terriers using two polymorphic minisatellites has shown that a significant reduction occurs in the number of alleles seen compared to an agglomerated population sample, correlating with the high level of inbreeding within this breed. Flanking DNA sequence and partial repeat sequence is presented for the most polymorphic minisatellite thus far identified, cCfaMP5. The variable region in this mini-satellite is similar to human minisatellites which show a distinct purine or pyrimidine strand bias.     

Characterization of tomato DNA clones with sequence similarity to human minisatellites 33.6 and 33.15

A tomato lambda genomic library was screened with the human minisatellites 33.6 and 33.15. Similar tomato sequences are estimated to occur on average every 4000 kb. In thirteen hybridizing clones characterized, the size of minisatellite arrays varied between 100 bp and 3 kb. The structure of the repetitive elements is complex as the human core sequence is interspersed with other elements. In three cases, sequences similar to the human minisatellites were part of a higher-order tandem repeat. The chromosomal position of these sequences was established by ascertaining linkage to previously mapped RFLP markers. In contrast to the human genome, no clustering of minisatellite loci was observed in tomato. The fingerprints generated by hybridizing tomato minisatellites to genomic DNA of a set of cultivars were, in two cases, more variable than those obtained with 33.6 or 33.15. Two of the characterized probes detected 4–8 alleles of a single locus, which displayed 10–15 times more polymorphism than random RFLP clones. Some minisatellites contain di- and tri-nucleotide microsatellite repeated motifs which may account for the high level of polymorphism detected with these clones.     

Characterization of Novel Minisatellite Repeat Loci in Atlantic Salmon (Salmo salar) and Their Phylogenetic Distribution

We present here the sequence and characterization of various minisatellite-like tandem repeat loci isolated from the genome of Atlantic salmon (Salmo salar). Their diversity of sequence and lack of core motifs common to minisatellites of other species suggest the presence of numerous and previously unidentified simple sequence repeat families in this salmonid. Evidence for their ubiquity was provided by screening of a salmon genomic library. Southern blot analysis of the phylogenetic distribution of a subset of the minisatellites found one sequence to be pervasive among vertebrates, others present only in Salmoninae or Salmonidae species, and one amplified only in Atlantic salmon. There is evidence for the positioning of microsatellite and minisatellite arrays in close proximity at many loci. Furthermore, one tandem repeat appears to have been inserted into the transposase coding region of a copy of the Tc1 transposon-like element recently identified in salmonids. Received: 9 October 1996 / Accepted: 20 May 1997     

A comparative genetic analysis of the Irish greyhound population using multilocus DNA fingerprinting, canine single locus minisatellites and canine microsatellites

Pairwise analysis of Hin fI/33·6 DNA fingerprints from a total of one hundred and fifty-three Irish greyhounds of known pedigree were used to determine band-share estimates of unrelated, first-degree and second-degree relationships. Forty-eight unrelated Irish greyhounds were used to determine allele frequencies for three single-locus minisatellites, and following a preliminary screen, eight of the most polymorphic tetra-nucleotide microsatellites from a panel of 15. The results indicated that both band-share estimates by DNA fingerprinting and microsatellite allele frequencies are highly effective in resolving parentage in this greyhound population, while single-locus minisatellites showed limited polymorphism and could not be used alone for routine parentage testing in this breed. The present study also demonstrated that, to obtain optimal resolution of parentage, sample sets of known pedigree status are required to determine the band-share distribution and/or microsatellite allele frequencies.     

Minisatellite polymorphism as a tool to distinguish closely related Lactococcus lactis strains

Genome plasticity is considered as a means for bacteria to adapt to their environment. Plasticity in tandem repeat sequences on bacterial genomes has been recently exploited to trace the epidemiology of pathogens. Here, we examine the utility of minisatellite (i.e., a repeat unit of six nucleotides or more) typing in non-pathogenic food bacteria of the species Lactococcus lactis. Thirty-four minisatellites identified on the sequenced L. lactis ssp. lactis strain IL1403 genome were first analyzed in 10 closely related ssp. lactis strains, as determined by randomly amplified polymorphic DNA (RAPD). The selected tandem repeats varied in length, percent identity between repeats, and locations. We showed that: (i) the greatest polymorphism was in orfs encoding exported proteins or in intergenic regions; (ii) two thirds of minisatellites were little- or non-variable, despite as much as 90% identity between tandem repeats; and (iii) dendrograms based on either RAPD or minisatellite analyses were similar. Seven minisatellites identified in this study are potentially useful for lactococcal typing. We then asked whether tandem repeats in L. lactis were stable upon very long-term (up to two years) storage. Despite large rearrangements previously reported in derivative strains, just one of 10 minisatellites tested underwent an alteration, suggesting that tandem repeat rearrangements probably occur during active DNA replication. We conclude that multiple locus minisatellite analysis can be a valuable tool to follow lactococcal strain diversity.     

A panel of VNTR markers in pigs

By cloning tandemly repeated sequences from the pig genome by use of non-porcine minisatellite probes for library screening, five novel polymorphic VNTR loci were isolated: three minisatellites and two satellite-like loci. Four of them could be mapped onto chromosomes by linkage analysis and/or in situ hybridization. They were assigned to Chromosomes (Chrs) 5, 6, 14, and 16. Physical mapping on both presumed satellites and on one of the minisatellites revealed that the former resided near or at the centromere and the latter towards the chromosome ends. The location of the minisatellite is of particular interest since, together with data on three other minisatellites previously isolated, it supports the idea that, as in humans, minisatellites may preferentially be subtelomeric also in pigs. Received: 23 August 1995 / Accepted: 5 March 1996     

Systematic cloning of human minisatellites from ordered array charomid libraries

We present a rapid and efficient method for the isolation of minisatellite loci from human DNA. The method combines cloning a size-selected fraction of human MboI DNA fragments in a charomid vector with hybridization screening of the library in ordered array. Size-selection of large MboI fragments enriches for the longer, more variable minisatellites and reduces the size of the library required. The library was screened with a series of multi-locus probes known to detect a large number of hypervariable loci in human DNA. The gridded library allowed both the rapid processing of positive clones and the comparative evaluation of the different multi-locus probes used, in terms of both the relative success in detecting hypervariable loci and the degree of overlap between the sets of loci detected. We report 23 new human minisatellite loci isolated by this method, which map to 14 autosomes and the sex chromosomes.     

Characterization of minisatellites in Arabidopsis thaliana with sequence similarity to the human minisatellite core sequence.

A strategy based on random PCR amplification was used to isolate new repetitive elements of Arabidopsis thaliana. One of the random PCR product analyzed by this approach contained a tandem repetitive minisatellite sequence composed of 33 bp repeated units. The genomic locus corresponding to this PCR product was isolated by screening a lambda genomic library. New related loci were also isolated from the genomic library by screening with a 14 mer oligonucleotide representing a region conserved among the different repeated units. Alignment of the consensus sequence for each minisatellite locus allowed the definition of an Arabidopsis thaliana core sequence that shows strong sequence similarities with the human core sequence and with the generalized recombination signal Chi of Escherichia coli. The minisatellites were tested for their ability to detect polymorphism, and their chromosomal position was established.     

Megasatellites: a peculiar class of giant minisatellites in genes involved in cell adhesion and pathogenicity in Candida glabrata

Minisatellites are DNA tandem repeats that are found in all sequenced genomes. In the yeast Saccharomyces cerevisiae, they are frequently encountered in genes encoding cell wall proteins. Minisatellites present in the completely sequenced genome of the pathogenic yeast Candida glabrata were similarly analyzed, and two new types of minisatellites were discovered: minisatellites that are composed of two different intermingled repeats (called compound minisatellites), and minisatellites containing unusually long repeated motifs (126-429 bp). These long repeat minisatellites may reach unusual length for such elements (up to 10 kb). Due to these peculiar properties, they have been named 'megasatellites'. They are found essentially in genes involved in cell-cell adhesion, and could therefore be involved in the ability of this opportunistic pathogen to colonize the human host. In addition to megasatellites, found in large paralogous gene families, there are 93 minisatellites with simple shorter motifs, comparable to those found in S. cerevisiae. Most of the time, these minisatellites are not conserved between C. glabrata and S. cerevisiae, although their host genes are well conserved, raising the question of an active mechanism creating minisatellites de novo in hemiascomycetes.