Distortion of quantitative genomic and expression hybridization by Cot-1 DNA: mitigation of this effect

Cross-hybridization of repetitive sequences in genomic and expression arrays is reported to be suppressed with repeat-blocking nucleic acids (C_ot-1 DNA). Contrary to expectation, we demonstrated that C_ot-1 also enhanced non-specific hybridization between probes and genomic targets. When added to target DNA, C_ot-1 enhanced hybridization (2.2- to 3-fold) to genomic probes containing conserved repetitive elements. In addition to repetitive sequences, C_ot-1 was found to be enriched for linked single copy (sc) sequences. Adventitious association between these sequences and probes distort quantitative measurements of the probes hybridized to desired genomic targets. Quantitative microarray hybridization studies using C_ot-1 DNA are also susceptible to these effects, especially for probes that map to genomic regions containing conserved repetitive sequences. Hybridization measurements with such probes are less reproducible in the presence of C_ot-1 than for probes derived from sc regions or regions containing divergent repeat elements, a finding with significant ramifications for genomic and expression microarray studies. We mitigated the requirement for C_ot-1 either by hybridizing with computationally defined sc probes lacking repeats or by substituting synthetic repetitive elements complementary to sequences in genomic probes.     

利用栽培稻C0t-1DNA和基因组DNA比较分析斑点野生稻和短药野生稻基因组

通过荧光原位杂交(FISH)利用来源于A基因组栽培稻的中高度重复序列C0t-1DNA和基因组DNA作为探针,对栽培稻、斑点野生稻和短药野生稻进行了比较基因组分析。结果发现C0t-1DNA杂交信号主要分布在这3种染色体的着丝粒、近着丝粒和端粒区域,在斑点野生稻染色体上的信号多于短药野生稻,与gDNA作为探针FISH的结果相一致,说明A和B基因组间的亲缘关系明显近于A和F基因组。确定了含有中高度重复序列的C0t-1DNA用于属内种间关系研究的可行性,并根据C0t-1DNA的FISH结果进行了染色体核型分析。     

Construction of repeat-free fluorescence in situ hybridization probes

FISH probes are generally made out of BAC clones with genomic DNA containing a variable amount of repetitive DNA that will need to be removed or blocked for FISH analysis. To generate repeat free (RF) Probes without loss in genomic coverage, a random library is made from BAC clones by whole-genome amplification (WGA). Libraries are denatured in the presence of excess C(0)t-1 DNA and allowed to re-anneal followed by digestion of all double-stranded elements by duplex-specific nuclease (DSN). Selective amplification of all elements not containing repetitive sequences is realized by a sequential amplification. The final RF products can be re-amplified and used as a stock for future probe production. The RF probes have a lower background, the signal intensity build up is faster and there is no need for blocking DNA. The signal to background ratio of the RF was higher as compared to repeat containing probes.     

自身基因组原位杂交揭示植物基因组重复DNA沿染色体的分布

重复DNA沿染色体的分布是认识植物基因组的组织和进化的要素之一。本研究采用一种改良的基因组原位杂交程序,对基因组大小和重复DNA数量不同的6种植物进行了自身基因组原位杂交(self-genomic in situ hybridization,self-GISH)。在所有供试物种的染色体都观察到荧光标记探针DNA的不均匀分布。杂交信号图型在物种间有明显的差异,并与基因组的大小相关。小基因组拟南芥的染色体几乎只有近着丝粒区和核仁组织区被标记。基因组相对较小的水稻、高粱、甘蓝的杂交信号分散分布在染色体的全长,但在近着丝粒区或近端区以及某些异染色质臂的分布明显占优势。大基因组的玉米和大麦的所有染色体都被密集地标记,并在染色体全长显示出强标记区与弱标记或不标记区的交替排列。此外,甘蓝染色体的所有近着丝粒区和核仁组织区、大麦染色体的所有近着丝粒区和某些臂中间区还显示了增强的信号带。大麦增强的信号带带型与其N-带带型一致。水稻自身基因组原位杂交图型与水稻Cot-1DNA在水稻染色体上的荧光原位杂交图型基本一致。研究结果表明,自身基因组原位杂交信号实际上反映了基因组重复DNA序列对染色体的杂交,因而自身基因组原位杂交技术是显示植物基因组中重复DNA聚集区在染色体上的分布以及与重复DNA相关联的染色质分化的有效方法。     

利用栽培稻C0t-1 DNA和基因组DNA比较分析宽叶野生稻基因组

利用AA染色体组栽培稻的中高度重复序列C0t-1 DNA和基因组DNA作为探针,通过荧光原位杂交技术对宽叶野生稻（Oryza latifolia）（CCDD染色体组）进行了比较基因组分析。结果显示,在宽叶野生稻染色体上,C0t-1 DNA的杂交信号没有基因组DNA的杂交信号明显;杂交信号主要分布在着丝粒、近着丝粒及端粒区域;随着洗脱严谨度的不同,杂交信号呈现出较高的种特异性。本研究以不同洗脱严谨度下的荧光原位杂交结果为依据,对宽叶野生稻进行的核型分析,可进一步提高稻属染色体识别的准确性。     

白菜rDNA及C0t-1DNA的荧光原位杂交及其核型分析

以早熟白菜苔为实验材料,从其基因组DNA中分离出C0t-1DNA并用生物素标记作探针,25SrDNA用地高辛标记作探针,对有丝分裂中期相染色体进行双色荧光原位杂交。每对染色体上均显示出了特定的C0t-1DNA荧光原位杂交带型,5对染色体上显示出了25SrDNA荧光原位杂交带型。双色荧光原位杂交证实了C0t-1DNA与25SrDNA二者具有一致的染色体位置特征,表明基于rDNA及C0t-1 DNA的荧光原位杂交核型分析技术,优于目前普遍采用的只基于rDNA的荧光原位杂交核型分析方法。结合C0t-1 DNA与25SrDNA的荧光原位杂交带型和传统的染色体的形态学标记分析方法及白菜已公布的基于rDNA分布的核型分析结果,创建了一个精确的白菜核型。     

玉米有丝分裂染色体上玉米BAC探针的FISH杂交体系的构建

本研究分别探讨了玉米和水稻基因组c 0t DNA对探针的封阻、杂交后洗脱的严谨度、杂交液中FAD的浓度变化对BAC-FISH杂交的影响;探讨了玉米BAC探针中重复序列含量对FISH信号的影响.初步形成了一套以玉米BAC探针在玉米有丝分裂染色体上进行FISH杂交的优化技术体系.结果表明,玉米基因组c 0t DNA对探针封阻的c 0t值应小于50;而降低杂交液中FAD浓度和适度控制杂交后洗脱的严谨度,尤其是使用水稻基因组的c 0t 100 DNA封阻探针重复序列对BAC-FISH杂交信号特异性的改善具有明显的效果;同时,验证了选择重复序列含量较少的玉米BAC作为FISH杂交的探针也是获得特异性杂交信号的重要条件.     

利用药用野生稻C基因组C_0t-1 DNA比较分析宽叶野生稻CCDD基因组

以来源于C基因组的药用野生稻的中高度重复序列C0t-1DNA为探针,在不同的洗脱严谨度下,通过荧光原位杂交对宽叶野生稻(CCDD)基因组进行了分析。结果发现,随着洗脱严谨度的调整,杂交信号呈现出较高的特异性,主要分布在着丝粒、近着丝粒及端粒区域。本文以宽叶野生稻的核型分析为基础,比较其与二倍体药用野生稻基因组的异同,从而进一步探讨野生稻的进化起源机制。     

Karyotyping of Brassica napus L. Based on C0t-1 DNA Banding by Fluorescence In Situ Hybridization

In order to precisely recognize and karyotype Brassica napus L. chromosomes, C0t-1 DNA was extracted from its genomic DNA, labeled with biotin-1 1-dUTP and in situ hybridized. The hybridized locations were detected by Cy3-conjugated streptavidin. Specific fluorescence in situ hybridization （FISH） signal bands were detected on all individual chromosome pairs. Each chromosome pair showed specific banding patterns. The B. napus karyotype has been constructed, for the first time, on the basis of both Cot-1 DNA FISH banding patterns and chromosome morphology.     

Highly repetitive component α and related alphoid DNAs in man and monkeys

The genomes of Old-World, New-World, and prosimian primates contain members of a large class of highly repetitive DNAs that are related to one another and to component DNA of the African green monkey by their sequence homologies and restriction site periodicities. The members, of this class of highly repetitive DNAs are termed the alphoid DNAs, after the prototypical member, component of the African green monkey which was the first such DNA to be identified (Maio, 1971) and sequenced (Rosenberg et al., 1978). The alphoid DNAs appear to be uniquely primate sequences. — From the restriction enzyme cleavage patterns and Southern blot hybridizations under different stringency conditions, the alphoid DNAs comprise multiple sequence families exhibiting varying degrees of homology to component DNA. They also share common elements in their restriction site periodicities (172 · n base-pairs), in the long-range organization of their repeating units, and in their banding behavior in CsCl and Cs₂SO₄ buoyant density gradients, in which they band within the bulk DNA as cryptic repetitive components. — In the three species from the Family Cercopithecidae examined, the alphoid DNAs represent the most abundant, tandemly repetitive sequence components, comprising about 24% of the African green monkey genome and 8 to 10% of the Rhesus monkey and baboon genomes. In restriction digests, the bulk of the alphoid DNAs among the Cercopithecidae appeared quantitatively reduced to a simple series of arithmetic segments based on a 172 base-pair (bp) repeat. In contrast with these simple restriction patterns, complex patterns were observed when human alphoid DNAs were cleaved with restriction enzymes. Detailed analysis revealed that the human genome contains multiple alphoid sequence families which differ from one another both in their repeat sequence organization and in their degree of homology to the African green monkey component DNA. — The finding of alphoid sequences in other Old-World primate families, in a New-World monkey, and in a prosimian primate attests to the antiquity of these sequences in primate evolution and to the sequence conservatism of a large class of mammalian highly repetitive DNA. In addition, the relative conservatism exhibited by these sequences may distinguish the alphoid DNAs from more recently evolved highly repetitive components and satellite DNAs which have a more restricted taxonomical distribution.     

Toward a molecular paleontology of primate genomes

Families of related, but nonidentical repetitive DNA sequences, termed the alphoid DNAs, have been identified and characterized in representative species from seven major primate Families. The sequences appear as old as the primate Order itself: they are found in a prosimian (lemur), in a New World monkey, and in all Old World primates examined, including man. The alphoid DNAs are uniquely primate sequences and they may represent the most abundant repetitive DNAs in the primate genome. — A classification scheme for two major families of alphoid DNAs is proposed that is based upon restriction enzyme analysis and Southern blotting with radioactive probes prepared from component DNA (Maio, 1971) and from the human EcoRI dimer sequences (Manuelidis, 1976). The family of alphoid DNAs that hybridizes readily with component is termed the HindIII family of alphoid DNAs. This family shows an almost universal distribution among present-day primates. The family of DNA sequences that hybridizes readily with the human EcoRI dimer probe is termed the EcoRI dimer family of alphoid DNAs. This family may be restricted to the great apes and man. The two probes permitted the discrimination of different, but related alphoid families in present-day primates. Multiple alphoid sequence families are found within the genomes of individual primates and the major primate taxa can be characterized by the representations of the various alphoid DNAs within their genomes. — An Appendix is presented (Brown et al., 1981) indicating that competition hybridization effects may influence the autoradiographic banding patterns, and hence, the interpretations of Southern filter-transfer hybridizations when dealing with related repetitive sequences such as the alphoid DNAs that are present in abundance in eukaryotic genomes.     

Genomic Subtraction Recovers Rye-Specific DNA Elements Enriched in the Rye Genome

Repetitive DNA sequence families have been identified in methylated relic DNAs of rye. This study sought to isolate rye genome-specific repetitive elements regardless of the level of methylation, using a genomic subtraction method. The total genomic DNAs of rye-chromosome-addition-wheat lines were cleaved to short fragments with a methylation-insensitive 4-bp cutter, MboI, and then common DNA sequences between rye and wheat were subtracted by annealing with excess wheat genomic DNA. Four classes of rye-specific repetitive elements were successfully isolated from both the methylated and non-methylated regions of the genome. Annealing of the DNA mixture at a ratio of the enzyme-restricted fragments:the sonicated fragments (1:3–1:5) was key to this success. Two classes of repetitive elements identified here belong to representative repetitive families: the tandem 350-family and the dispersed R173 family. Southern blot hybridization patterns of the two repetitive elements showed distinct fragments in methylation-insensitive EcoO109I digests, but continuous smear signals in the methylation-sensitive PstI and SalI digests, indicating that both of the known families are contained in the methylated regions. The subtelomeric tandem 350-family is organized by multimers of a 380-bp-core unit defined by the restriction enzyme EcoO109I. The other two repetitive element classes had new DNA sequences (444, 89 bp) and different core-unit sizes, as defined by methylation-sensitive enzymes. The EcoO109I recognition sites consisting of PyCCNGGPu-multi sequences existed with high frequency in the four types of rye repetitive families and might be a useful tool for studying the genomic organization and differentiation of this species.     

莲C_0t-1 DNA的荧光原位杂交分析

为分析中国莲C_0t-1 DNA在其中期染色体上的分布,从中国莲基因组DNA中分离出C_0t-1 DNA,将基因组和所分离的C_0t-1 DNA用生物素标记后作探针,对中国莲染色体进行原位杂交。杂交结果用耦联有荧光素Cy3的生物素抗体检测,发现在每对染色体上均显示出特定的荧光原位杂交带。同时分析了FISH和GISH信号分布的异同。基于C_0t-1 DNA荧光原位杂交带型及染色体型,构建了中国莲核型。     

莲C0t-1 DNA的荧光原位杂交分析

为分析中国莲Cot-1DNA在其中期染色体上的分布,从中国莲基因组DNA中分离出Cot-1DNA,将基因组和所分离的Cot-1DNA用生物素标记后作探针,对中国莲染色体进行原位杂交。杂交结果用耦联有荧光素Cy3的生物素抗体检测,发现在每对染色体上均显示出特定的荧光原位杂交带。同时分析了FISH和GISH信号分布的异同。基于Cot-1DNA荧光原位杂交带型及染色体型,构建了中国莲核型。     

The evolution of repetitive DNA sequences in sea urchins.

Molecular hybridization of nuclear DNAs has been employed to study the evolution of the repetitive DNA sequences in four species of sea urchin. The data show that relative to S. purpuratus there has been approximately 0.1% sequence divergence per million years in the repetitive DNA sequences of S. droebachiensis, S. franciscanus, and L. pictus. These results confirm that repetitive DNA sequences are strongly conserved during evolution. However, comparison of the extent of base pair mismatch in the repetitive DNA heteroduplexes formed at Cot 20 with those formed at Cot 200 during the hybridization of S. purpuratus and L. pictus DNAs reveals that highly repetitive sequences of sea urchins may diverge more rapidly than do the more moderately repetitive sequences.     

Genetic mobility of plasmids of Streptomyces erythraeus

Grossing of S. erythraeus 4 with S. erythraeus 1 resulted in transfer of genetic elements from strain 4 to strain 1 as evidence by the 20 and 18 kb fragments in the experiments on DNA-DNA hybridization. The presence of the genetic elements in strain 1 was the cause of plasmid pSE 21 mobility. In strain 6, a derivative of S. erythraeus 1 plasmid pSE 21 was accompanied by other extrachromosomal DNAs characterized by high instability. During storage of the strain at a temperature of 4 degrees C for more than 1 or 2 months the number of the plasmid pSE 21 copies decreased. When the strain was stored for longer periods (6 months or more) the plasmid DNA was not detectable even with the DNA-DNA hybridization procedure. The results of hybridization of a fraction of the extrachromosomal DNA of S. erythraeus 6, the Bam HIB fragment of plasmid pSE 21 with the total DNA of strains 1, 4, 5, 6 and BTCC 2 of S. erythraeus and hybridization of DNA of plasmid pSE 21 with the total DNA of S. erythraeus 6 and 1 showed that (1) strains 1, 5 and BTCC 2 had the same hybridization patterns, (2) the other extrachromosomal DNAs present in the fraction were homologous with the Bam HIA fragment of plasmid pSE 21, (3) chromosomes of strains 1, 4, 5, 6 and BTCC 2 of S. erythraeus also contained DNA homologous to the plasmid Bam HIA fragment. It was suggested that plasmid pSE 21 could be used as a basis for constructing the integrative vector for S. erythraeus.     

Rearrangement of repeated DNA sequences during development of macronucleus in Tetrahymena thermophila.

Three clones of non-repetitive sequences and six clones containing repetitive sequences were obtained from micronuclear DNA of Tetrahymena thermophila. All the non-repetitive and three repetitive sequences had the same organization in micro- and macronuclear DNAs as revealed by blot hybridization. On the other hand, the remaining three clones with repetitive sequences had apparently different organization in the two nuclear DNAs. All these repetitive sequences showed a smear on the blot in addition to a number of discrete bands when micronuclear DNA was digested with EcoR I. In macronuclear DNAs, these sequences invariably became one or two bands and the smear disappeared. We conclude that, when a macronucleus develops from a micronucleus, the non-repetitive sequences amplify by more than 20 times with relatively few rearrangement, whereas some selected portions of repeated and/or repeat-contiguous sequences are amplified with rather extensive reorganization.     

用于目标序列染色体定位的镜鲤BAC-FISH体系构建

为了构建用于镜鲤(Cyprinus carpio var. specularis)特定基因组序列染色体定位的实验体系, 在细菌人工染色体(Bacterial Artificial Chromosome, BAC)文库筛选池中对已知短序列基因组片段进行PCR扩增, 筛选出包含目标序列的BAC克隆, 提取BAC质粒进行缺刻平移标记制备探针, 开展荧光原位杂交(Fluorescence in situ hybridization, FISH)实验。通过对染色体片前处理、BAC质粒探针制备、C0t-1 DNA封闭基因组重复序列、预杂交、荧光染料选择、信号放大等一系列实验条件和方法的探索优化, 成功实现了目标序列在镜鲤有丝分裂中期染色体上的定位。定位对象既包括在染色体上有单一位点的序列, 如斑马鱼微卫星标记Z6884和Z4268, 也包括在染色体上有多个位点的重复序列, 如黄河鲤性别相关标记CCmf1。来自斑马鱼同一条染色体上的两个微卫星标记被分别定位于镜鲤不同染色体上, 为鲤鱼染色体数目加倍的进化假设提供了一项直接实验证据, 同时将现有遗传连锁图谱与染色体对应起来, 可作为染色体识别和细胞遗传学图谱构建的依据。黄河鲤性别相关重复序列被定位于不少于四条染色体上, 为性别决定相关基因的筛查提供了研究线索。这一BAC-FISH实验体系将成为鲤细胞遗传学图谱构建、基因组进化和比较基因组学研究中的重要研究工具。       

SNTG1, the gene encoding γ1-syntrophin: a candidate gene for idiopathic scoliosis

Idiopathic scoliosis (IS) affects approximately 2%–3% of the population and has a heritable component. The genetics of this disorder are complex. Here, we describe a family in which a pericentric inversion of chromosome 8 co-segregates with IS. We have used fluorescence in situ hybridization to identify cloned DNAs that span the breakpoints on the two arms of the chromosome. We have identified a bacterial artificial chromosome (BAC) of 150 kb that crosses the q-arm breakpoint and a BAC of 120 kb that crosses the p-arm breakpoint. The complete genomic DNA sequence of these BACs has been analyzed to identify candidate genes and to localize further the precise breakpoints. This has revealed that the p-arm break does not interrupt any known gene and occurs in a region of highly repetitive sequence elements. On the q-arm, the break occurs between exons 10 and 11 of the -1 syntrophin (SNTG1) gene. Syntrophins are a group of cytoplasmic peripheral membrane proteins that associate directly with dystrophin, the Duchenne muscular dystrophy gene; 1-syntrophin has been shown to be a neuronal cell-specific protein. Mutational analysis of SNTG1 exons in 152 sporadic IS patients has revealed a 6-bp deletion in exon 10 of SNTG1 in one patient and a 2-bp insertion/deletion mutation occurring in a polypyrimidine tract of intronic sequence 20 bases upstream of the SNTG1 exon 5 splice site in two patients. These changes were not seen in a screen of 480 control chromosomes. Genomic DNAs from seven affected individuals within the family of a patient carrying the 6-bp deletion were typed to determine whether the alteration co-segregated with IS. The deletion was only observed in five out of these seven individuals. Thus, although genetic heterogeneity or multiple alleles cannot be ruled out, the 6-bp deletion does not consistently co-segregate with the disease in this family.Electronic Supplementary Material Supplementary material is available in the online version of this article at