Analysis of repetitive element DNA methylation by MethyLight

Repetitive elements represent a large portion of the human genome and contain much of the CpG methylation found in normal human postnatal somatic tissues. Loss of DNA methylation in these sequences might account for most of the global hypomethylation that characterizes a large percentage of human cancers that have been studied. There is widespread interest in correlating the genomic 5-methylcytosine content with clinical outcome, dietary history, lifestyle, etc. However, a high-throughput, accurate and easily accessible technique that can be applied even to paraffin-embedded tissue DNA is not yet available. Here, we report the development of quantitative MethyLight assays to determine the levels of methylated and unmethylated repeats, namely, Alu and LINE-1 sequences and the centromeric satellite alpha (Satα) and juxtacentromeric satellite 2 (Sat2) DNA sequences. Methylation levels of Alu, Sat2 and LINE-1 repeats were significantly associated with global DNA methylation, as measured by high performance liquid chromatography, and the combined measurements of Alu and Sat2 methylation were highly correlative with global DNA methylation measurements. These MethyLight assays rely only on real-time PCR and provide surrogate markers for global DNA methylation analysis. We also describe a novel design strategy for the development of methylation-independent MethyLight control reactions based on Alu sequences depleted of CpG dinucleotides by evolutionary deamination on one strand. We show that one such Alu-based reaction provides a greatly improved detection of DNA for normalization in MethyLight applications and is less susceptible to normalization errors caused by cancer-associated aneuploidy and copy number changes.     

Characterization of a highly repetitive family of DNA sequences in the mouse.

A large proportion (0.5-1%) of total mouse DNA is cleaved by Bam HI into fragments whose size is about 500 base pairs. A cloned member of this repetitive family of DNA sequences (BAM5 family) was sequenced by the dideoxy chain termination procedure and shown to contain 507 base pairs. The sequence exhibited no unusual or remarkable features. Repetitive sequences complementary to the cloned BAM5 fragment were found in rat DNA, but not in feline or human DNA. Restriction mapping suggested that many BAM5 sequences were components of much larger repetitive DNAs which were scattered throughout the mouse genome. The BAM5 sequences within the larger repetitive DNAs did not appear to be arranged tandemly or as members of scrambled tandem repeats. RNA homologous to the cloned BAM5 sequence was detected in cultured mouse cells, but not in cultured rat cells.     

Linearization and integration of DNA into cells preferentially occurs at intrinsically curved regions from human LINE-1 repetitive element.

A bent DNA library was constructed from human genomic DNA, from which a new clone belonging to the human LINE-1 sequence family was isolated and characterized. This clone, with a length of 378 base pairs and termed HBC-1 (human bent clone-1), contained an intrinsically occurring curved DNA structure. By permutation analysis, the center of curvature of this fragment was mapped onto the nucleotide position 886 from the 5' terminus of the complete LINE-1 sequence. Reporter plasmids, which contain HBC-1, were effectively integrated into human chromosome, indicating that the bent DNA structure provides a preferential donor site for the integration of human LINE-1 sequences. The present finding may provide an explanation as to why some inactivated LINE-1 sequences on human chromosomes carry the deletion at their 5' termini.     

Characterization of repetitive DNA in rye (Secale cereale)

Nuclear DNA of rye (Secale cereale), a plant species with a relatively large genome (i.e., 18 pg diploid), has been characterized by determination of its content in repetitive sequences, buoyant density, and thermal denaturation properties. The reassociation kinetics of rye DNA reveals the presence of 70 to 75% repeated nucleotide sequences which are grouped into highly (Cot 1) and intermediately repetitive (Cot 1–100) fractions. On sedimentation in neutral CsCl gradients, native, high molecular weight DNA forms an almost symmetrical band of density 1.702 g/cm³. The highly repetitive DNA (Cot 1), on the other hand, is separated into two distinct peaks; the minor component has a density of 1.703 g/cm³ corresponding to that of a very rapidly reassociating fraction (Cot 0.01) which comprises 10 to 12% of the rye genome. The latter DNA contains segments which are repeated 6×10⁵ to 6×10⁶ times. The major peak of the Cot 1 fraction shows a density of 1.707 g/cm³ and consists of fragments repeated about 3.7×10⁴ times. The intermediately repetitive DNA is much more heterogeneous than the Cot 1 fraction and has a low degree of repetition of the order of 8.5×10². The melting behavior of the Cot 1 fraction reveals the presence of a high degree of base pairing (i.e., 7% mismatching). When native rye DNA is resolved into fractions differing in GC content by hydroxyapatite thermal column chromatography and these fractions are analyzed for the presence of repetitive sequences, it is observed that the highly redundant DNA (Cot 1) is mostly located in the fraction denaturing between 80° and 90°C. This result suggests that highly repetitive rye DNA occurs in a portion of the genome which is neither very rich in AT nor in GC.     

A new repetitive element of the CR1 family downstream of the chicken vitellogenin gene.

We have analyzed a repetitive DNA sequence found in the 3'-flanking region of the chicken vitellogenin gene. By its sequence, the repetitive DNA has been identified as a hitherto unreported member of the chicken CR1 family of repetitive elements. The CR1 sequence displays the structural characteristics of a long terminal repeat located at the 3' end of an avian retrovirus. The CR1 element lies 2.2 kb downstream of the vitellogenin gene and 'points' away from the gene rather than toward it. In this respect, this element differs from other CR1 repeats. The CR1 element is embedded in a region showing changes in chromatin structure implying a potential role for this sequence in determining the structural state of the local chromatin.     

Integration of a vector containing a repetitive LINE-1 element in the human genome.

Mammalian cells contain numerous nonallelic repeated sequences, such as multicopy genes, gene families, and repeated elements. One common feature of nonallelic repeated sequences is that they are homeologous (not perfectly identical). Our laboratory has been studying recombination between homeologous sequences by using LINE-1 (L1) elements as substrates. We showed previously that an exogenous L1 element could readily acquire endogenous L1 sequences by nonreciprocal homologous recombination. In the study presented here, we have investigated the propensity of exogenous L1 elements to be involved in a reciprocal process, namely, crossing-overs. This would result in the integration of the exogenous L1 element into an endogenous L1 element. Of over 400 distinct integration events analyzed, only 2% involved homologous recombination between exogenous and endogenous L1 elements. These homologous recombination events were imprecise, with the integrated vector being flanked by one homologous and one illegitimate junction. This type of structure is not consistent with classical crossing-overs that would result in two homologous junctions but rather is consistent with one-sided homologous recombination followed by illegitimate integration. Contrary to what has been found for reciprocal homologous integration, the degree of homology between the exogenous and endogenous L1 elements did not seem to play an important role in the choice of recombination partners. These results suggest that although exogenous and endogenous L1 elements are capable of homologous recombination, this seldom leads to crossing-overs. This observation could have implications for the stability of mammalian genomes.     

FBXO44 promotes DNA replication-coupled repetitive element silencing in cancer cells

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Characterization of CARE-1: Candida albicans repetitive element-1

A middle repetitive DNA element, Candida albicans repetitive element-1 (CARE-1) has been isolated from the pathogenic yeast C. albicans. CARE-1 appears to be species-specific and constitutes approx. 0.045% of total C. albicans DNA, or a reiteration frequency of about two to twelve copies per haploid genome. The CARE-1 element has been detected on several C. albicans chromosomes separated by field-inversion gel electrophoresis, suggesting that the element is dispersed. Interstrain variation was observed in the number and distribution of hybridizing bands. The element is well conserved, since no nucleotide (nt) heterogeneity was observed when the sequences of two CARE-1 family members isolated from two different chromosomes (A and B) of C. albicans were compared. CARE-1 possesses 467 bp and is characterized by several stretches of A's and T's, short direct repeats and shows no significant homology to any known nt sequence.     

Cloning and characterization of a centromere-specific repetitive DNA element from Sorghum bicolor

 A 823-bp Sau3AI fragment (pSau3A10) was subcloned from a sorghum bacterial artificial chromosome (BAC) clone, 13I16, that contains DNA sequences specific to the centromeres of grass species. Sequence analysis showed that pSau3A10 consists of six copies of an approximately 137-bp monomer. The six monomers were organized into three dimers. The monomers within the dimers shared 62–72% homology and the dimers were 79–82% homologous with each other. Fluorescence in situ hybridization (FISH) analysis indicated that the Sau3A10 family is present only in the centromeres of sorghum chromosomes. Sequencing, Southern hybridization, and Fiber-FISH analyses indicated that the Sau3A10 family is tandemly arranged and is present in uninterrupted stretches of up to at least 81 kb of DNA. Slot-blot analysis estimated that the Sau3A10 family constitutes 1.6–1.9% of the sorghum genome. The long stretches of Sau3A10 sequences were interrupted by other centromeric DNA elements. Southern analysis indicated that the Sau3A10 sequence is one of the most abundant DNA families located in sorghum centromeres and is conserved only in closely related sorghum species. Methylation experiments indicated that the cytosine of the CG sites in sorghum centromeric regions is generally methylated. The structure and organization of the Sau3A10 family shared similarities with centromeric DNA repeats in other eukaryotic species. It is suggested that the Sau3A10 family is probably an important part of sorghum centromeres. Received: 11 November 1997 / Accepted: 17 November 1997     

大豆疫霉菌一个DNA指纹分析重复序列探针的鉴定

【目的】大豆疫霉菌指纹分析的建立和黑龙江与新疆大豆疫霉菌群体的群体遗传分析。【方法】利用生物信息学方法寻找大豆疫霉菌(Phytophthora sojae)的中度重复序列,并对黑龙江和新疆大豆疫霉菌进行DNA指纹分析。【结果】分析得到一个中度重复序列,定名为PS1227。Southern blot分析表明PS1227在大豆疫霉菌基因组中约有34条可辨的介于1.5-23kb之间的杂交条带,其中21个杂交条带在49个供试菌系中表现多态性。单游动孢子分析表明PS1227指纹特征在病菌无性生殖阶段表现稳定。利用PS1227标记,本实验发现采自黑龙江HP4002、SY6和GJ0105菌系分别与新疆的DW303、71228和71222菌系具有完全相同的指纹特征。【结论】获得一个可用于大豆疫霉菌流行学和群体生物学研究的指纹分析序列PS1227,在分子水平证实了新疆大豆疫霉菌可能由黑龙江传入。     

The MER3 DNA helicase catalyzes the unwinding of holliday junctions

The MER3 protein of Saccharomyces cerevisiae is required for crossover in meiosis and has been suggested to act at the initiation of homologous pairing and the resolution of Holliday junctions. The purified MER3 protein is a DNA helicase that translocates along single-stranded DNA in the 3' to 5' direction displacing annealed DNA fragments. Here, MER3 was found to be able to unwind various double-stranded DNA (dsDNA) substrates, including a 30-bp dsDNA with a 20-nucleotide 3'-overhang, a 30-bp dsDNA with a 20-nucleotide 5'-overhang, a 50-bp dsDNA with blunt ends, and a Holliday junction with 25-bp arms, each of which had a blunt end. Efficient unwinding of the 3'-overhang substrate appeared to initiate by the binding of MER3 to the 3' single-stranded tail in a reaction that required six or more unpaired bases. Unwinding of the blunt end and 5'-overhang substrates appeared to initiate at the blunt ends of these substrates. Unwinding of the Holliday junction was more efficient than the unwinding of the blunt and 5'-overhang substrates and was influenced by Mg(2+) concentrations that cause changes in the structure of the junction. Possible roles for Holliday junction unwinding in meiotic crossover are discussed.     

Primate evolution of a human chromosome 1 hypervariable repetitive element

Summary The clone designated hMF #1 represents a clustered DNA family, located on chromosome 1, consisting of tandem arrays displaying a monomeric length of 40 bp and a repetition frequency of approximately 7×10³ copies per haploid genome. The sequence hMF #1 reveals multiple restriction fragment length polymorphisms (RFLPs) when human genomic DNA is digested with a variety of 4–6-bp recognition sequence restriction enzymes (i.e., Taq I, Eco RI, Pst I, etc.). When hamster and mouse genomic DNA was digested and analyzed, no cross-species homology could be observed. Further investigation revealed considerable hybridization in the higher primates (chimpanzee, gorilla, and orangutan) as well as some monkey species.The evolutionary relationship of this repetitive DNA sequence, found in humans, to that of other primates was explored using two hybridization methods: DNA dot blot to establish copy number and Southern DNA analysis to examine the complexity of the RFLPs. Homology to the hMF #1 sequence was found throughout the suborder Anthropoidea in 14 ape and New and Old World monkey species. However the sequence was absent in one species of the suborder Prosimii. Several discrepancies between established evolutionary relationships and those predicted by hMF #1 exist, which suggests that repetitive elements of this type are not reliable indicators of phylogenetic branching patterns. The phenomenon of marked diversity between sequence homologies and copy numbers of dispersed repetitive DNA of closely related species has been observed inDrosophila mice,Galago, and higher primates. We report here a similar phenomenon for a clustered repeat that may have originated at an early stage of primate evolution.     

Characterization of the formae speciales of Fusarium oxysporum causing wilts of cucurbits by DNA fingerprinting with nuclear repetitive DNA sequences.

The genetic relatedness of five formae speciales of Fusarium oxysporum causing wilts of cucurbit plants was determined by DNA fingerprinting with the moderately repetitive DNA sequences FOLR1 to FOLR4. The four FOLR clones were chosen from a genomic library made from F. oxysporum f. sp. lagenariae 03-05118. Total DNAs from 50 strains representing five cucurbit-infecting formae speciales, cucumerinum, melonis, lagenariae, niveum, and momordicae, and 6 strains of formae speciales pathogenic to other plants were digested with EcoRV and hybridized with 32P-labeled FOLR probes. The strains were clearly distinguishable at the formae specialis level on the basis of FOLR DNA fingerprints. Fifty-two fingerprint types were detected among the 56 strains by using all FOLR probes. These probes were used to infer phylogenetic relationships among the DNA fingerprint types by the unweighted pair group method using averages and parsimony analysis. The fingerprint types detected in each of the formae speciales cucumerinum, lagenariae, niveum, and momordicae were grouped into a single cluster. However, two different genetic groups occurred in the formae specialis melonis. The two groups also differed in pathogenicity: one group caused wilts of muskmelon and oriental melon, while the second was pathogenic only to muskmelon. The fingerprint types of different formae speciales pathogenic to plants other than cucurbits were distinguishable from one another and from the fingerprints of the cucurbit-infecting strains. These results suggest that the cucurbit-infecting formae speciales are intraspecific variants distinguishable at the DNA level and in their host range.     

Characterization and assessment of an avian repetitive DNA sequence as an icterid phylogenetic marker.

The first tandemly repeated sequence examined in a passerine bird, a 431-bp PstI fragment named pMAT1, has been cloned from the genome of the brown-headed cowbird (Molothrus ater). The sequence represents about 5-10% of the genome (about 4 x 10(5) copies) and yields prominent ethidium bromide stained bands when genomic DNA cut with a variety of restriction enzymes is electrophoresed in agarose gels. A particularly striking ladder of fragments is apparent when the DNA is cut with HinfI, indicative of a tandem arrangement of the monomer. The cloned PstI monomer has been sequenced, revealing no internal repeated structure. There are sequences that hybridize with pMAT1 found in related nine-primaried oscines but not in more distantly related oscines, suboscines, or nonpasserine species. Little sequence similarity to tandemly repeated PstI cut sequences from the merlin (Falco columbarius), saurus crane (Grus antigone), or Puerto Rican parrot (Amazona vittata) or to HinfI digested sequence from the Toulouse goose (Anser anser) was detected. The isolated sequence was used as a probe to examine DNA samples of eight members of the tribe Icterini. This examination revealed phylogenetically informative characters. The repeat contains cutting sites from a number of restriction enzymes, which, if sufficiently polymorphic, would provide new phylogenetic characters. Sequences like these, conserved within a species, but variable between closely related species, may be very useful for phylogenetic studies of closely related taxa.