Molecular analysis of N6-methyladenine patterns in Tetrahymena thermophila nuclear DNA.

We have cloned two DNA fragments containing 5'-GATC-3' sites at which the adenine is methylated in the macronucleus of the ciliate Tetrahymena thermophila. Using these cloned fragments as molecular probes, we analyzed the maintenance of methylation patterns at two partially and two uniformly methylated sites. Our results suggest that a semiconservative copying model for maintenance of methylation is not sufficient to account for the methylation patterns we found during somatic growth of Tetrahymena. Although we detected hemimethylated molecules in macronuclear DNA, they were present in both replicating and nonreplicating DNA. In addition, we observed that a complex methylation pattern including partially methylated sites was maintained during vegetative growth. This required the activity of a methylase capable of recognizing and modifying sites specified by something other than hemimethylation. We suggest that a eucaryotic maintenance methylase may be capable of discriminating between potential methylation sites to ensure the inheritance of methylation patterns.     

Sequence organisation in nuclear DNA from Physarum polycephalum: Genomic organisation of DNA segments containing foldback sequences

DNA clones containing foldback sequences, derived from Physarum polycephalum nuclear DNA, can be classified according to their pattern of hydridisation to Southern blots of genomic DNA. One group of DNA clones map to unique DNA loci when used as a probe to restriction digests of Physarum nuclear DNA. These cloned segments appear to contain dispersed repetitive sequence elements located at many hundreds of sites in the genome. Similar patterns of hybridisation are generated when these cloned DNA probes are annealed to DNA restriction fragments of genomic DNA obtained from a number of different Physarum strains, indicating that no detectable alteration has occurred at these genomic loci subsequent to the divergence of the strains as a result of the introduction or deletion of mobile genetic elements. However, deletion of segments of some cloned DNA fragments occurs following their propagation in Escherichia coli. A second, distinct group of clones are shown to be derived from highly methylated segments of Physarum DNA which contain very abundant repetitive sequences with regular, though complex, arrangements of restriction sites at their various genomic locations. It is suggested that these DNA segments contain clustered repetitive sequence elements. The results lead to the conclusion that foldback elements in Physarum DNA are located in segments of the genome which display markedly different patterns of sequence organisation and degree of DNA methylation.     

Macronuclear DNA of Tetrahymena thermophila exists as defined subchromosomal-sized molecules.

Using the method of orthogonal-field-alternation gel electrophoresis, we have resolved the macronuclear DNA of Tetrahymena thermophila into a series of distinct bands. Using electrode switching intervals ranging from 10 to 70 seconds we have resolved DNA bands ranging in size from about 21 kb up to and beyond the size of yeast chromosomes VII and XV. Hybridization of Southern blots from these gels to both unique and repetitive DNA sequences shows that the macronuclear genome of T. thermophila has a precise organization. The unique sequences tested each hybridize to only one band of macronuclear DNA and the hybridization patterns seem to be identical in several inbred strains examined.     

Modeling DNA methylation in a population of cancer cells

Little is known about how human cancers grow because direct observations are impractical. Cancers are clonal populations and the billions of cancer cells present in a visible tumor are progeny of a single transformed cell. Therefore, human cancers can be represented by somatic cell ancestral trees that start from a single transformed cell and end with billions of present day cancer cells. We use a genealogical approach to infer tumor growth from somatic trees, employing haplotype DNA methylation pattern variation, or differences between specific CpG sites or "tags," in the cancer genome. DNA methylation is an epigenetic mark that is copied, with error, during genome replication. At our tags, neutral copy errors in DNA methylation appear to occur at random, and much more frequently than sequence copy errors. To reconstruct a cancer tree, we sample and compare human colorectal genomes within small geographic regions (a cancer fragment), between fragments on the same side of the tumor, and between fragments from opposite tumor halves. The combined information on both physical distance and epigenetic distance informs our model for tumor ancestry. We use approximate Bayesian computation, a simulation-based method, to model tumor growth under a variety of evolutionary scenarios, estimating parameters that fit observed DNA methylation patterns. We conclude that methylation patterns sampled from human cancers are consistent with replication errors and certain simple cancer growth models. The inferred cancer trees are consistent with Gompertzian growth, a well-known cancer growth pattern.     

DNA ANALYSIS OF EUKARYOTIC ALGAL SPECIES1

Plastid genomes of algae resemble those of terrestrial plants in form, size, and rate of nucleotide sequence change. They are circular and range in size from 73 kilobases (kb) to over 400 kb. Their many copies per cell can compose >15% of total cell DNA. Mitochondrial genomes, like plastid genomes, are present in high copy number in preparations of total algal cell DNA. Almost all known algal mitochondrial DNA genomes are relatively small, <50 kb; in some species they are linear, whereas in others they are circular. One of the persistent perplexities for phycologists is the question of what relationship two clones or two groups of organisms bear to each other. Several relatively simple techniques can reveal whether or not two organisms belong to the same clone. Total mitochondrial genome size can be compared directly between isolates, although identity in size does not necessarily mean identity in sequence. Restriction endonuclease digestion combined with probing permits comparison of DNA fragment patterns to see if there is identity or near identity between two samples. This methodology can be applied both to organelle genomes and to nuclear genomes. So far, restriction endonucleases cleave plastid and mitochondrial DNA of organisms belonging to the same gene pool into nearly identical fragment patterns, whereas organisms nearly or totally incapable of interbreeding display patterns wherein ? 50% of restriction fragments differ in position on an agarose gel after electrophoresis. Thus, organelle genomes may be the first choice for comparing both total size and restriction endonuclease fragment patterns to obtain an indication of whether two organisms are closely related. This methodology can be applied both to organisms in which interbreeding is easy to test and to the many algae in which homothallism or lack of sexual clones has precluded standard breeding analyses. With further data on variability levels within and between fertile populations, it may be possible to state with confidence whether a sample of morphologically similar organisms shares a common gene pool, even if their breeding cannot be manipulated experimentally.     

Fast algorithms for large-scale genome alignment and comparison

We describe a suffix-tree algorithm that can align the entire genome sequences of eukaryotic and prokaryotic organisms with minimal use of computer time and memory. The new system, MUMmer 2, runs three times faster while using one-third as much memory as the original MUMmer system. It has been used successfully to align the entire human and mouse genomes to each other, and to align numerous smaller eukaryotic and prokaryotic genomes. A new module permits the alignment of multiple DNA sequence fragments, which has proven valuable in the comparison of incomplete genome sequences. We also describe a method to align more distantly related genomes by detecting protein sequence homology. This extension to MUMmer aligns two genomes after translating the sequence in all six reading frames, extracts all matching protein sequences and then clusters together matches. This method has been applied to both incomplete and complete genome sequences in order to detect regions of conserved synteny, in which multiple proteins from one organism are found in the same order and orientation in another. The system code is being made freely available by the authors.     

Characterization of a novel satellite DNA sequence from Flying Dragon (Poncirus trifoliata)

Repetitive sequences constitute a significant component of most eukaryotic genomes, and the isolation and characterization of repetitive DNA sequences provide an insight into the organization of the genome of interest. Here, we report the isolation and the molecular analysis and methylation status of a novel tandemly organized repetitive DNA sequence from the genome of Poncirus trifoliata. Digestion of P. trifoliata DNA with Afa I produced a prominent fragment of approximately 400 bp. Southern blotting analysis of genomic DNA digested with the same enzyme revealed a ladder composed of DNA fragments that are multimers of the 400-bp Afa I band, indicating that the repetitive DNA is arrayed in tandem. This suggests that Afa I isolated a novel satellite that we have called Poncirus trifoliata satellite DNA 400 (PN400). This satellite composes 25% of the genome and it is also present in lemon, sour orange and kumquat. Analysis of the methylation status demonstrated that the cytosines in CCGG sequences in this satellite were methylated.     

Organization of mitochondrial DNA in normal and texas male sterile cytoplasms of maize

Recombinant DNA and hybridization techniques have been used to compare the organization of mitochondrial DNA (mtDNA) from normal (N) and Texas male sterile (T) cytoplasms of maize. Bam H1 restriction fragments of normal mtDNA were cloned and used in molecular hybridizations against Southern blots of Bam H1 digested N and T mtDNA. Fifteen of the 35 fragments were conserved in both N and T as indicated by hybridization to comigrating bands in their restriction patterns. Only three fragments produced autoradiographs whose differences could reasonably be attributed to single changes in the cleavage site of the enzyme while approximately half (17/35) of the clones resulted in more complicated differences between N and T. The autoradiographs produced by these 17 clones indicated multiple cleavage site changes and/or sequence rearrangements of the mtDNA. Patterns of six of these 17 clones indicated partial duplication of the sequence and two showed variation in the intensity of hybridization between N and T, which may be related to the molecular heterogeneity phenomenon found in maize mitochondrial genomes. The large proportion of changes observed between N and T mtDNA indicates that rearrangements may have played an important role in the evolution of the maize mitochondrial genome.     

Repetitive Sequences in Plant Nuclear DNA:Types,Distribution, Evolution and Function

Repetitive DNA sequences are a major component of eukaryotic genomes and may account for up to 90% of the genome size. They can be divided into minisatellite, microsatellite and satellite sequences. Satellite DNA sequences are considered to be a fast-evolving component of eukaryotic genomes, comprising tandemly-arrayed, highly-repetitive and highly-conserved monomer sequences. The monomer unit of satellite DNA is 150–400 base pairs(bp) in length.Repetitive sequences may be species- or genus-specific, and may be centromeric or subtelomeric in nature. They exhibit cohesive and concerted evolution caused by molecular drive, leading to high sequence homogeneity. Repetitive sequences accumulate variations in sequence and copy number during evolution, hence they are important tools for taxonomic and phylogenetic studies, and are known as ‘‘tuning knobs' ' in the evolution. Therefore, knowledge of repetitive sequences assists our understanding of the organization, evolution and behavior of eukaryotic genomes. Repetitive sequences have cytoplasmic, cellular and developmental effects and play a role in chromosomal recombination. In the post-genomics era, with the introduction of next-generation sequencing technology, it is possible to evaluate complex genomes for analyzing repetitive sequences and deciphering the yet unknown functional potential of repetitive sequences.     

Comparative analyses of DNA methylation and sequence evolution using Nasonia genomes

The functional and evolutionary significance of DNA methylation in insect genomes remains to be resolved. Nasonia is well situated for comparative analyses of DNA methylation and genome evolution, since the genomes of a moderately distant outgroup species as well as closely related sibling species are available. Using direct sequencing of bisulfite-converted DNA, we uncovered a substantial level of DNA methylation in 17 of 18 Nasonia vitripennis genes and a strong correlation between methylation level and CpG depletion. Notably, in the sex-determining locus transformer, the exon that is alternatively spliced between the sexes is heavily methylated in both males and females, whereas other exons are only sparsely methylated. Orthologous genes of the honeybee and Nasonia show highly similar relative levels of CpG depletion, despite ~190 My divergence. Densely and sparsely methylated genes in these species also exhibit similar functional enrichments. We found that the degree of CpG depletion is negatively correlated with substitution rates between closely related Nasonia species for synonymous, nonsynonymous, and intron sites. This suggests that mutation rates increase with decreasing levels of germ line methylation. Thus, DNA methylation is prevalent in the Nasonia genome, may participate in regulatory processes such as sex determination and alternative splicing, and is correlated with several aspects of genome and sequence evolution.     

Covalent genomic DNA modification patterns revealed by denaturing gradient gel blots

Several approaches are used to survey genomic DNA methylation patterns, including Southern blot, PCR, and microarray strategies. All of these methods are based on the use of methylation-sensitive isoschizomer restriction enzyme pairs and/or sodium bisulfite treatment of genomic DNA. They have many limitations, including PCR bias, lack of comprehensive assessment of methylated sites, labor-intensive protocols, and/or the need for expensive equipment. Since the presence of 5-methylcytosine alters the melting properties of DNA molecules, denaturing gradient gel blots (DGG blots), a gene scanning technique which detects differences in DNA fragments based on differential melting behavior, were used to examine genomic modification patterns in normal tissues. Variations in melting behavior, observed as restriction fragment melting polymorphisms (RFMPs), were detected in various tissues from single individuals in all human and mouse genes tested, suggesting the presence of widespread differential cell type-specific DNA modification. Additional DGG blot experiments comparing genomic DNA to unmethylated cloned DNA suggested that the melting variants were most likely caused by DNA methylation differences. The results suggest that the use of DGG blots can provide a comprehensive and rapid method for comparing complex in vivo DNA modification patterns in normal adult somatic cells.     

Hybridization monitor: a method for identifying differences between complex genomes

We have developed a method to identify and amplify differential fragments between two complex genomes. This technique, named hybridization-monitored genome differential analysis (HMDA), incorporates a monitor system into a PCR-based solid subtraction hybridization that tracks the entire hybridization process. This is achieved by monitoring the subtraction progress using PCR analysis of the conserved sequence of 18S rDNA in the tester sample after each round of subtraction. Homologous fragments can then be eliminated when bound to the driver DNA immobilized on a solid membrane. The hybridization continues until the conserved DNA sequence of 18S rDNA can no longer be detected, and most of the unbound DNA fragments left in the liquid were mainly the tester-specific fragments, thus greatly decreasing the complexity of DNA template of PCR amplification, increasing the amplification efficiency of differences accordingly, and ensuring high positive efficiency and coverage across the tester genome. We have applied the technique in a comparison between the genomes of Saccharomyces cerevisiae and Schizosaccharomyces pombe, which are two completely sequenced organisms. Results indicated that 95% of the subtracted clones have been confirmed to be different to the driver analyzed using the BLASTN homology alignment. With this technique, 240-fold enrichment of differences is obtained, and the coverage of the difference is up to 79%. These results indicate that HMDA can efficiently identify sequences that differ between two complex genomes.     

Detection and mapping of homologous, repeated and amplified DNA sequences by DNA renaturation in agarose gels.

A new molecular hybridization approach to the analysis of complex genomes has been developed. Tracer and driver DNAs were digested with the same restriction enzyme(s), and tracer DNA was labeled with 32P using T4 DNA polymerase. Tracer DNA was mixed with an excess amount of driver, and the mixture was electrophoresed in an agarose gel. Following electrophoresis, DNA was alkali-denatured in situ and allowed to reanneal in the gel, so that tracer DNA fragments could hybridize to the driver only when homologous driver DNA sequences were present at the same place in the gel, i.e. within a restriction fragment of the same size. After reannealing, unhybridized single-stranded DNA was digested in situ with S1 nuclease. The hybridized tracer DNA was detected by autoradiography. The general applicability of this technique was demonstrated in the following experiments. The common EcoRI restriction fragments were identified in the genomes of E. coli and four other species of bacteria. Two of these fragments are conserved in all Enterobacteriaceae. In other experiments, repeated EcoRI fragments of eukaryotic DNA were visualized as bands of various intensity after reassociation of a total genomic restriction digest in the gel. The situation of gene amplification was modeled by the addition of varying amounts of lambda phage DNA to eukaryotic DNA prior to restriction enzyme digestion. Restriction fragments of lambda DNA were detectable at a ratio of 15 copies per chicken genome and 30 copies per human genome. This approach was used to detect amplified DNA fragments in methotrexate (MTX)-resistant mouse cells and to identify commonly amplified fragments in two independently derived MTX-resistant lines.