Global methylation screening in the Arabidopsis thaliana and Mus musculus genome: applications of virtual image restriction landmark genomic scanning (Vi-RLGS)

Understanding the role of ‘epigenetic’ changes such as DNA methylation and chromatin remodeling has now become critical in understanding many biological processes. In order to delineate the global methylation pattern in a given genomic DNA, computer software has been developed to create a virtual image of restriction landmark genomic scanning (Vi-RLGS). When using a methylation- sensitive enzyme such as NotI as the restriction landmark, the comparison between real and in silico RLGS profiles of the genome provides a methylation map of genomic NotI sites. A methylation map of the Arabidopsis genome was created that could be confirmed by a methylation-sensitive PCR assay. The method has also been applied to the mouse genome. Although a complete methylation map has not been completed, a region of methylation difference between two tissues has been tested and confirmed by bisulfite sequencing. Vi-RLGS in conjunction with real RLGS will make it possible to develop a more complete map of genomic sites that are methylated or demethylated as a consequence of normal or abnormal development.     

Spot Mapping on the Standard Profile of Restriction Landmark Genomic Scanning (RLGS) of Sorted Chromosome 20 Using Methylation-Insensitive Enzyme

We established the spot mapping system on a restriction landmark genomic scanning (RLGS) profile using sorted chromosome as RLGS material. In this mapping system, we can mapped RLGS spots physically, regardless of their polymorphism, using methylation-insensitive enzymes in all RLGS steps. Here, we report that we identified 28 spots derived from human chromosome 20 on an RLGS profile, and that number was in good agreement with the number predicted from the length of the chromosome 20.     

A New Tool for the Rapid Cloning of Amplified and Hypermethylated Human DNA Sequences from Restriction Landmark Genome Scanning Gels

Restriction landmark genome scanning (RLGS) is an effective genome-scanning technique capable of identifying DNA amplification and aberrant DNA methylation. Previously published methods for the cloning of human DNA fragments from RLGS gels have been successful only for high-copy-number fragments (repetitive elements or DNA amplifications). We present here the first technique capable of efficiently cloning single-copy human DNA fragments ("spots") identified in RLGS profiles. This technique takes advantage of a plasmid-based, human genomic DNA, NotI/EcoRV boundary library. The library is arrayed in microtiter plates. When clones from a single plate are pooled and mixed with genomic DNA, the resultant RLGS gel is a normal profile with a defined set of spots showing enhanced intensity for that particular plate. This was performed for a set of 32 plates as well as their pooled rows and columns. Thus, we have mapped individual RLGS spots to exact plate, row, and column addresses in the library and have thereby obtained immediate access to these clones. The feasibility of the technique is demonstrated in examples of cloning methylated DNA fragments identified in human breast tumor and testicular tumor RLGS profiles and in the cloning of an amplified DNA fragment identified in a human medulloblastoma RLGS profile.     

Differentiation of trophoblast lineage is associated with DNA methylation and demethylation.

Our previous study has shown that the placenta and kidney had different genomic methylation patterns regarding CpG island loci detected by restriction landmark genomic scanning (RLGS). To investigate whether differentiation involves changes in DNA methylation, we analyzed the rat Rcho-1 cell line, which retains trophoblast cell features and differentiates from stem cells into trophoblast giant cells in vitro. By RLGS, a total of 1,232 spots were identified in the Rcho-1 stem and differentiated giant cells. Four spots (0.3%) were detected only in giant cells, implying that the loci were originally methylated, but became demethylated during differentiation. Another four spots (0.3%) were detected only in stem cells, implying that these loci, originally unmethylated, became methylated during differentiation. DNAs from three loci that became methylated during differentiation were cloned and sequenced. All showed high homologies with expressed sequence tags (ESTs) or with genomic DNA of other species, suggesting that these loci are biologically important. Thus, the eight differentially methylated loci should be good tools to study epigenetic modification specific to differentiation of trophoblast giant cells.     

Direct Determination of NotI Cleavage Sites in the Genomic DNA of Adult Mouse Kidney and Human Trophoblast Using Whole-Range Restriction Landmark Genomic Scanning

Restriction landmark genomic scanning (RLGS) is a method forvisualizing restriction landmarks, employing direct labelingof restriction sites of genomic DNA and high-resolution two-dimensionalelectrophoresis. We determined the conditions for both the firstand second dimensions of RLGS that define all of the restrictionfragments which carry the NotI landmark. Using this system,we determined the number of cleavable NotI sites of genomicDNA from the mouse kidney (C57BL/6) and from the human placenta.The mouse and human genomes were cleaved at 2,380±80sites (4,760±160 spots) and 3,240±110 sites (6,480±220spots), respectively with NotI.     

Comparison of DNA methylation patterns among mouse cell lines by restriction landmark genomic scanning.

Restriction landmark genomic scanning (RLGS) is a novel method which enables us to simultaneously visualize a large number of loci as two-dimensional gel spots. By this method, the status of DNA methylation can efficiently be determined by monitoring the appearance or disappearance of spots by using a methylation-sensitive restriction enzyme. In the present study, using RLGS with NotI, we examined, in comparison with a brain RLGS profile, the status of DNA methylation of more than 900 loci among three types of mouse cell lines: the embryonal carcinoma cell line P19, the stable mesenchymal cell line 10T1/2, and our established neuroepithelial (EM) cell lines. We found that the relative numbers of RLGS spots which appeared were less than 3.3% of those surveyed in all cell lines examined. However, 5 to 14% of spots disappeared, the numbers increasing with an increase in the length of the culture period, and many spots were commonly lost in 10T1/2 and in three EM cell lines. Thus, for these cell lines, many more spots disappeared than appeared. However, the numbers of spots disappearing and appearing were well balanced, and the ratio in P19 cells was almost equal to that in liver cells in vivo. These RLGS experimental observations suggested that permanent cell lines such as 10T1/2 are hypermethylated and that our newly established EM cell lines are also becoming heavily methylated at common loci. On the other hand, methylation and demethylation seem to be balanced in P19 cells in a manner similar to that in in vivo liver tissue.     

Analysis of CpG islands of trophoblast giant cells by restriction landmark genomic scanning

Rat trophoblast giant cells each contain at least 100 times more genomic DNA per nucleus than diploid cells. This unusual phenomenon appears to be of interest in relation to the molecular mechanism of cell differentiation and gene expression in the placenta. In the present study, we analyzed the CpG islands of trophoblast giant cells by restriction landmark genomic scanning (RLGS) using the methylation-sensitive landmark enzymes, Not I and Bss HII. More than 1,000 and 1,900 spots were detected by RLGS using Not I and Bss HII, respectively, in the placental junctional zone, where more than 90% of genomic DNA is present in the cells with higher DNA content. Of these, 97% (1,009 spots) and 99% (1,911 spots) of the spots found in the junctional zone showed an identical pattern and identical intensity with those of diploid cell controls, for which genomic DNA was extracted from the labyrinth zone and maternal kidney. Therefore, the giant cells are basically polyploid. More importantly, 24 tissue-specific spots were detected by RLGS using Not I. Subsequent cloning and sequencing of four typical spots of the genomic DNA confirmed that these DNA fragments contained abundant CpG dinucleotides and showed characteristics of CpG islands. Of these 24 spots, there were ten spots specific for the placenta, and three of them were specific for the junctional zone, indicating that methylation status of CpG islands in the placental tissue differed between the junctional zone and labyrinth zone. These results suggest that multiple rounds of endoreduplication and modification of CpG islands by cytosine methylation occur during the differentiation process of giant cells. Dev. Genet. 22:132–140, 1998. © 1998 Wiley-Liss, Inc.     

Restriction landmark genomic scanning

Restriction landmark genomic scanning (RLGS) is a method to detect large numbers of restriction landmarks in a single experiment. It is based on the concept that restriction enzyme sites can serve as landmarks throughout a genome. RLGS uses direct end-labeling of the genomic DNA digested with a rare-cutting restriction enzyme and high-resolution two-dimensional electrophoresis. Compared with the conventional gene-detection technologies, such as Southern blot analysis and PCR, RLGS has the following advantages even though it needs specially designed instruments: high-efficiency scanning capacity, scanning extensibility by using alternate restriction enzyme combinations, applicability to any organism, a spot intensity that reflects the copy number of restriction landmarks, and the ability, by using a methylation-sensitive enzyme, to screen the methylated state of genomic DNA. The RLGS protocol can be accomplished in 5 days to 2 weeks.     

In silico restriction landmark genome scanning analysis of Xanthomonas oryzae pathovar oryzae MAFF 311018

We have developed a restriction landmark genome scanning (RLGS) system in silico, involving two-dimensional electrophoretic analysis of DNA by computer simulation that is based on the availability of whole-genome sequences for specific organisms. We applied the technique to the analysis of the Xanthomonas oryzae pathovar oryzae (Xoo) MAFF 311018, which causes bacterial blight in rice. The coverage that was found to be achievable using RLGS in silico, as a percentage of the genomic regions that could be detected, ranged from 44.5% to 72.7% per image. However, this reached a value of 96.7% using four images that were obtained with different combinations of landmark restriction enzymes. Interestingly, the signal intensity of some of the specific spots obtained was significantly lower than that of other surrounding spots when MboI, which cleaves unmethylated 5'-GATC-3' sites, was used. DNA gel blot analysis with both DNA adenine methylase (Dam)-sensitive and -insensitive isoschizomers (MboI and Sau3AI) revealed that Dam-mediated DNA adenine methylation had indeed occurred at these particular sites. These results suggest that a significant portion of the 5'-GATC-3' sites within the Xoo genome is stably methylated by Dam.     

DNA adenine methylation changes dramatically during establishment of symbiosis

The DNA adenine methylation status on specific 5'-GANTC-3' sites and its change during the establishment of plant-microbe interactions was demonstrated in several species of alpha-proteobacteria. Restriction landmark genome scanning (RLGS), which is a high-resolution two dimensional DNA electrophoresis method, was used to monitor the genomewide change in methylation. In the case of Mesorhizobium loti MAFF303099, real RLGS images obtained with the restriction enzyme MboI, which digests at GATC sites, almost perfectly matched the virtual RLGS images generated based on genome sequences. However, only a few spots were observed when the restriction enzyme HinfI was used, suggesting that most GANTC (HinfI) sites were tightly methylated and specific sites were unmethylated. DNA gel blot analysis with the cloned specifically unmethylated regions (SUMs) showed that some SUMs were methylated differentially in bacteroids compared to free-living bacteria. SUMs have also been identified in other symbiotic and parasitic bacteria. These results suggest that DNA adenine methylation may contribute to the establishment and/or maintenance of symbiotic and parasitic relationships.     

Application of the RLGS image analysis tool (RAT) to the construction of a genetic linkage map of recombinant inbred strain SMXA

The construction of a genetic linkage map is the first, fundamental step to analyze the genetic properties of any organism. For this purpose, the restriction landmark genome scanning method (RLGS) can be used and has been shown to have high productivity in various genetic analyses. However, construction of a genetic linkage map by the RLGS method is laborious, because hundreds of spots must be scored, usually by visual observation. In order to reduce human involvement in the data processing, we developed an image analysis software, RAT (RLGS Analysis Tool). We evaluated its accuracy and feasibility by comparing the parental distribution patterns of RLGS spots obtained by RAT and by human observation, using Syrian hamster strain backcross progeny. We then used RAT to construct a genetic linkage map of the recombinant inbred strain SMXA. We were able to obtain 121 progenitor strain-specific spots that were assigned to a specific chromosome. Received: 1 December 1998 / Accepted: 29 January 1999     

A Genetic Linkage Map of the Mouse Using Restriction Landmark Genomic Scanning (Rlgs)

We have developed a multiplex method of genome analysis, restriction landmark genomic scanning (RLGS) that has been used to construct genetic maps in mice. Restriction landmarks are end-labeled restriction fragments of genomic DNA that are separated by using high resolution, two-dimensional gel electrophoresis identifying as many as two thousand landmark loci in a single gel. Variation for several hundred of these loci has been identified between laboratory strains and between these strains and Mus spretus. The segregation of more than 1100 RLGS loci has been analyxed in recombinant inbred (RI) strains and in two separate interspecific genetic crosses. Genetic maps have been derived that link 1045 RLGS loci to reference loci on all of the autosomes and the X chromosome of the mouse genome. The RLGS method can be applied to genome analysis in many different organisms to identify genomic loci because it used end-labeling of restriction landmarks rather than probe hybridization. Different combinations of restriction enzymes yield different sets of RLGS loci providing expanded power for genetic mapping.     

Linkage map of Syrian hamster with restriction landmark genomic scanning

We have constructed the linkage map with precise genetic analysis of the Syrian hamster, Mesocricetus auratus, according to the restriction landmark genomic scanning (RLGS) spot mapping method. Although only 3.2–6.6% of the total RLGS spots between the two strains, ACN and BIO 14.6, showed genetic variance, 572 loci were found to be polymorphic. Out of 569 RLGS loci and 3 other loci, 531 were mapped with the backcross (ACN × BIO 14.6) F₁× BIO 14.6. The cumulative map was 1111.6 cM, indicating that the spots/loci are located throughout the genome at 1.94 cM intervals on average. Thus, RLGS provides us with a rapid tool to construct the genetic map of any species, even if it has less genetic variation. Received: 15 July 1996 / Accepted: 25 September 1996     

High-Speed Positional Cloning Based on Restriction Landmark Genome Scanning

Restriction landmark genome scanning (RLGS) was developed as a method of genome analysis that is based on the concept that restriction enzyme sites can be used as landmarks. In this article, we demonstrate how this method can be used for the systematic, successful positional cloning of mouse mutantreelergene. The major advantage of the RLGS method is that it allows the scanning of several thousand spots/loci throughout the genome with one RLGS profile. High-speed positional cloning based on the RLGS method includes (1) high-speed construction of a linkage map (RLGS spot mapping), (2) high-speed detection of RLGS spot markers tightly linked to the mutant phenotype (RLGS spot bombing method), and (3) construction of YAC contigs covering the region where tightly linked spot markers are located (RLGS-based YAC contig mapper). We introduced a series of these procedures by using them to positionally clone thereelergene. High-speed construction of the whole genetic map and spots/loci (less than 1 cM) within the closest flanking markers is demonstrated. The RLGS-based YAC contig mapper also efficiently yielded the YAC physical contig map of the target region. Finally, we cloned thereelergene, which is the causal gene for the perturbation of the three-dimensional brain architecture due to the abnormal migration of neuroblasts inreelermouse. Since the RLGS method itself can be used for any organism, we conclude that the total RLGS-based positional cloning system can be used to identify any mutant gene of any organism.     

Restriction landmark genomic scanning (RLGS) in fungi

RLGS is a technique to detect DNA polymorphism using restriction sites as landmarks. It identifies the landmarks through direct end-labeling, two-dimensional electrophoresis and autoradiography, giving a profile with many spots to allow the scanning of numerous DNA loci. We successfully performed the technique on fungi using isolates ofColletotrichum acutatum andC. gloeosporioides in anamorphic Ascomycotina,Rhizopus oryzae in Zygomycotina,Phytophthora nicotianae in Mastigomycotina (or Oomycota) andRhizoctonia solani in anamorphic basidiomycotina. RLGS of total genomic DNA digested with three restriction enzymes,Not, I,EcoR V andMbo I, reproducibly gave specific profiles of ca. 400 to 1.600 spots for each isolate. A polymorphic spot appearing to reflect a genetic difference between the twoColletotrichum species was found in the profiles of the isolates. No other common spots were found in any combination of isolates of the twoColletotrichum species, and thus the other spots on the profiles were regarded as unique to each isolate. These results indicated that RLGS could be applied, as a powerful fingerprinting technique based on genetic information from the whole genomic DNA, to search for useful DNA markers for taxonomic and genomic studies on many fungal species.     

Adaptation of Restriction Landmark Genomic Scanning (RLGS) to plant genome analysis

Restriction Landmark Genomic Scanning (RLGS) profiles, which are based on the concept of using restriction enzyme sites as landmarks and are generated by two-dimensional gel electrophoresis, were applied to the analysis of plant genomes. This study identified landmark enzymes in RLGS profiles of rice, tobacco, and arabidopsis, because the success of RLGS analysis depends on finding useful landmarks and these are the most frequently studied higher plants. As the results demonstrate, in each plant RLGS analysis various landmark enzymes were identified as useful landmark enzymes. However, the number of spots in a single profile was smaller than in mouse RLGS profiles and differed remarkably in the various plants. In addition, we demonstrate the optimal electrophoresis conditions and a convenient spot-cloning method.     

Improved strain distribution patterns of SMXA recombinant inbred strains by microsatellite markers.

To develop SMXA recombinant inbred (RI) strains as more valuable genetic resources, 302 microsatellite (Mit) loci were added to the strain distribution patterns (SDP) reported previously. The improved SDP were constructed in a total of 1085 loci containing 484 Mit markers, 571 restriction landmark genomic scanning (RLGS) spot markers and 30 others. This substantially improved SDP can be freely accessed on our homepage (http://www.med.nagoya-u.ac.jp/sisetu/SDP.htm).     

Genetic profile of the SMXA recombinant inbred mouse strains revealed with restriction landmark genomic scanning

We have applied the restriction landmark genomic scanning (RLGS) method to the SMXA recombinant inbred (RI) mouse strain set to reveal its detailed genetic profile. A total of 663 polymorphic RLGS spot loci were identified, 576 of which were assigned to chromosomes. Strain distribution patterns (SDPs) at 55 microsatellite marker loci were also obtained. As a result, the total number of loci with distinct SDPs on chromosomes increased to 400. These loci were dispersed on all chromosomes, except for the Chromosome (Chr) Y, and effectively covered the genome with an average spacing of 4 cM. The SMXA RI strain set, hereby, would be of value for genetic study. Received: 20 February 1998 / Accepted: 19 May 1998     

Genome-wide analysis of DNA methylation status of CpG islands in embryoid bodies, teratomas, and fetuses

Differentiation of embryonic stem (ES) cells into embryoid bodies (EBs) provides an in vitro system for the study of early lineage determination during mammalian development. We have previously reported that there are 247 CpG islands that potentially have tissue-dependent and differentially methylated regions (T-DMRs). This provided evidence that the formation of DNA methylation patterns at CpG islands is a crucial epigenetic event underlying mammalian development. Here we present an analysis by the restriction landmark genomic scanning (RLGS) using NotI as a landmark enzyme of the genome-wide methylation status of CpG islands of ES cells and EBs and of teratomas produced from ES cells. These results are considered in relation to the methylation status of CpG islands of genomic DNA from normal fetus (10.5 dpc) and adult tissues. We have prepared a DNA methylation panel that consists of 259 T-DMRs and includes novel T-DMRs that are distinctly methylated or unmethylated in the teratomas. The DNA methylation pattern was complex and differed for the ES cells, EBs, and teratomas, providing evidence that differentiation of cells involves both de novo DNA methylation as well as demethylation. Comparison of the numbers of T-DMRs, that were differentially methylated or unmethylated among the cells and tissue types studied, revealed that the teratomas were the most epigenetically different from ES cells. Thus, analysis of the DNA methylation profiles prepared in this study provides new insights into the differentiation of ES cells and development of fetus, EB, teratoma, and somatic tissues.