A genetic linkage map of the MSM Japanese wild mouse strain with restriction landmark genomic scanning (RLGS)

A high-resolution genetic map of the Mus musculus molossinus (MSM) Japanese wild mouse strain was constructed with restriction landmark genomic scanning (RLGS) and compared with that of the laboratory strain C3H. MSM is phylogenetically 1 million years apart from common laboratory mouse strains and is distinctly resistant to chemical carcinogenesis. Since it exhibits frequent genetic polymorphisms with laboratory mice but can still be easily crossed with laboratory strains, hybrids between MSM and carcinogen-sensitive laboratory mouse strains provide excellent materials for analysis of modifier genes and genetic changes during carcinogenesis. We have generated MSM backcross progeny with the C3H strain, which is extremely sensitive to hepatocarcinogenesis, to construct the present map. RLGS profiles with two combinations of restriction enzymes (NotI–PvuII–PstI, NotI–PstI–PvuII) yielded more than 2000 spots each. The polymorphism rate was about 39.2%, and of a total of 1732 polymorphic spot loci identified, 1371 could be assigned to specific chromosomes by comparison with 79 microsatellite marker loci. Thus, 1450 loci, on all chromosomes except for Y, effectively mapped 90% of the genome (1431.7 cM length). Although some spots might be derived from the same NotI site, each NotI site potentially generating two fragments, the presence of at least 515 loci groups with different progeny distribution patterns dispersed through the genome with an average spacing of 3 cM, means that this genetic map should be useful for analysis of various biological phenomena, including carcinogenesis and ontogenesis, at the gene level. Received: 25 August 1999 / Accepted: 20 December 1999     

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.     

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.     

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.     

Application of the RLGS Method to Large-Size Genomes Using a Restriction Trapper

We developed a method for producing restriction landmark genomicscanning (RLGS) profiles of large size genomes, such as thoseof higher plants or amphibians using a restriction trapper.Use of the conventional RLGS method is limited to genomes smallerthan 3 x 10⁹ bp, because the larger genomic DNAs, especiallythose of more than 1 x 10¹⁰ bp, produce high background dueto incorporation of radioactivity at non-specifically damagedsites. Our new method reduces the background levels by reducinggenome complexity to 1/200–1/300 using a purificationstep to enrich DNA fragments carrying specific restriction landmarksat their ends using a restriction trapper. This step makes itpossible to obtain RLGS patterns of larger genomes. Our paperdescribes the practical application for the RLGS method usinga restriction trapper with the pine tree genome (3 x 10¹⁰ bp/haploidgenome; Pinus koraiensis Sieb. et Zucc.) as an example.     

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.     

Nucleotide Sequence Surrounding the Locus Marker D21S246 on Human Chromosome 21

Most ofthe human Not I linking clones identified to date areconsidered to be derived from CpG islands because ofthe recognitionsequence of this enzyme, and CpG islands have been reportedto be located around the 5' regions of genes. As a pilot study,we determined the complete nucleotide sequence (41,924 bp) ofa human cosmid clone (LL21NC02Q7A10) containing the marker D21S246originating from a Not I linking clone. As a result of sequenceanalysis, we successfully mapped and revealed the genomic genestructure for KIAA0002 previously reported as a cDNA clone.This gene consists of 15 exons and was shown to exist at theD21S246 locus on human chromosome 21q21.3–q22.1. Theseresults demonstrated that genomic marker-anchored DNA sequencingis a useful approach for the human genome project.     

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.     

Reproducible Alterations of DNA Methylation at a Specific Population of CpG Islands during Blast Formation of Peripheral Blood Lymphocytes

We investigated the changes in the methylation patterns of CpGislands associated with blast formation of human peripheralblood lymphocytes activated by anti-CD3 and interleukin-2 (IL-2),using restriction landmark genomic scanning with a methylation-sensitiverestriction enzyme (RLGS-M) system. Of about 2,100 Not I spot/lociwhich were analyzed, only 10 showed changes, whereas drasticchanges have been observed in cases of malignant and SV40 transformation.These changes were highly reproducible for samples from boththe same and different individuals. Even the timing of the changesafter cultivation was the same. Thus, we concluded that at leastthe genomic DNA methylation state in vivo was essentially retainedin T blast cells activated in vitro by induction with IL-2 andanti-CD3, which are commonly used in biological experimentsas well as clinical diagnosis and therapy.     

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     

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.     

Epigenetics: application of virtual image restriction landmark genomic scanning (Vi-RLGS)

Restriction landmark genomic scanning (RLGS) is a powerful method for the systematic detection of genetic mutations in DNA length and epigenetic alteration due to DNA methylation. However, the identification of polymorphic spots is difficult because the resulting RLGS spots contain very little target DNA and many non-labeled DNA fragments. To overcome this, we developed a virtual image restriction landmark genomic scanning (Vi-RLGS) system to compare actual RLGS patterns with computer-simulated RLGS patterns (virtual RLGS patterns). Here, we demonstrate in detail the contents of the simulation program (rlgssim), based on the linear relationship between the reciprocal of mobility plotted against DNA fragment length and Vi-RLGS profiling of Arabidopsis thaliana.     

Molecular analysis of human Chromosome 16 cosmid clones containing NotI sites

To test the feasibility of using cloned NotI sites as markers for physical mapping, we have screened for cosmid clones spanning the NotI sites on human Chromosome (Chr) 16. Fluorescence in situ hybridization analysis of these clones confirms the previously reported cluster of NotI sites on 16p13.3. Methylation status of the cloned NotI sites on genomic DNA was established by hybridization of the cosmids to Southern blots containing EcoRI and EcoRI/NotI digest of genomic DNA. These results indicated that four of six clones included in our study can be used as linking clones for physical mapping. Two clones have NotI sites which are not cleavable in the cell lines tested. In one clone, the NotI site exists as an isolated rare-cutting restriction enzyme site, whereas in the other clone the NotI site appears to be island-related.     

Determination of Gardnerella vaginalis Genome Size by Pulsed-Field Gel Electrophoresis

The chromosomal DNA of four strains of Gardnerella vaginaliswere digested with rare cutting restriction enzymes and analyzedby pulsed-field gel electrophoresis (PFGE). The four strainsstudied were two clinical isolates (GVP 004 & GVP 007) andtwo American Type Culture Collection strains (ATCC 14018 &ATCC 14019). The restriction enzyme SfiI generated two DNA fragmentsof about 0.6 Mb and 1.1 Mb in all four strains giving a G. vaginalisgenome size of about 1.7 Mb. A similar genome size was calculatedutilizing two more GC-rich sequence specific restriction endonucleases,NotI and AscI. When digested with AscI, the chromosomal DNAof all four strains gave rise to 11 to 12 DNA fragments rangingbetween 0.01 Mb to 0.43 Mb. DNA from the two clinical isolateswere digested by NotI (yielding 7 to 9 fragments), while theDNA from the two ATCC strains were resistant to NotI digestion.In contrast to the clinical isolates, DNA from the two ATCCstrains gave an identical profile for all restriction endonucleasestested. From double digestion experiments, the two SfiI sitescould be localized on two AscI fragments. From these PFGE studies,it is concluded that the G. vaginalis genome is a circular DNAthat ranges between 1.67 Mb and 1.72 Mb in size.     

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.     

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.     

EagI andNotI linking clones from human chromosomes 11 and Xp

EagI and NotI linking libraries were prepared in the lambda vector, EMBL5, from the mouse-human somatic cell hybrid 1W1LA4.9, which contains human chromosomes 11 and Xp as the only human component. Individual clones containing human DNA were isolated by their ability to hybridise with total human DNA and digested with SalI and EcoRI to identify the human insert size and single-copy fragments. The mean (± SD) insert sizes of the EagI and NotI clones were 18.3 ± 3.2 kb and 16.6 ± 3.6 kb, respectively. Regional localisation of 66 clones (52 EagI, 14 NotI) was achieved using a panel of 20 somatic cell hybrids that contained different overlapping deletions of chromosomes 11 or Xp. Thirty-nine clones (36 EagI, 3 NotI) were localised to chromosome 11; 17 of these were clustered in 11q13 and another nine were clustered in 11q14–q23.1. Twenty-seven clones (16 EagI, 11 NotI) were localised to Xp and 10 of these were clustered in Xp11. The 66 clones were assessed for seven different microsatellite repetitive sequences; restriction fragment length polymorphisms for five clones from 11q13 were also identified. These EagI and NotI clones, which supplement those previously mapped to chromosome 11 and Xp, should facilitate the generation of more detailed maps and the identification of genes that are associated with CpG-rich islands. Received: 27 December 1995 / Revised: 30 January 1996     

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.     

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.