Restriction landmark genome scanning.

Restriction landmark genome scanning (RLGS) method is a high-resolution two-dimensional electrophoresis system for analyses of the whole genome DNA which is including methylation status. It has been used for cloning genes of model animals and human genomes, detection of imprinted genes, and genome-wide methylation research in cancer. The conventional RLGS detected both polymorphism and methylated NotI sites between samples. Here, we have developed improved RLGS method with isoschizomer restriction enzymes such as MspI and HpaII to specifically detect methylated sites, using differential sensitivity of the restriction enzymes to methylated sequences. Recently, by using the genome database information, the RLGS spot sites were efficiently identified by this improved method. Then, genome methylation sites of Arabidopsis were mapped, and a unique inheritance was detected in methylated gene in rice. Now, epigenetic research becomes easy with the improved RLGS and it also can be applied for animal genome. Therefore, RLGS method is useful to explore for novel epigenetic phenomenon.

The application of restriction landmark genome scanning method for surveillance of non-mendelian inheritance in f(1) hybrids.

We analyzed inheritance of DNA methylation in reciprocal F(1) hybrids (subsp. japonica cv. Nipponbare x subsp. indica cv. Kasalath) of rice (Oryza sativa L.) using restriction landmark genome scanning (RLGS), and detected differing RLGS spots between the parents and reciprocal F(1) hybrids. MspI/HpaII restriction sites in the DNA from these different spots were suspected to be heterozygously methylated in the Nipponbare parent. These spots segregated in F(1) plants, but did not segregate in selfed progeny of Nipponbare, showing non-Mendelian inheritance of the methylation status. As a result of RT-PCR and sequencing, a specific allele of the gene nearest to the methylated sites was expressed in reciprocal F(1) plants, showing evidence of biased allelic expression. These results show the applicability of RLGS for scanning of non-Mendelian inheritance of DNA methylation and biased allelic expression.

Inheritance and alteration of genome methylation in F1 hybrid rice.

We analyzed the inheritance of DNA methylation in the first filial generation(F1) hybrid of Oryza sativa L. ("Nipponbare"x"Kasalath") by restriction landmark genome scanning (RLGS). Most parental RLGS spots were found in the F1, but eight spots (4%) showed abnormal inheritance: seven of the eight spots were missing in the F1, and one was newly detected in the F1. Here we show demethylation at restriction enzyme sites in the F1. We also found a candidate site of stable heterozygous methylation in the genome. These results show the applicability of the RLGS method for analysis of the inheritance and alteration of methylation in F1 hybrid plants.

OsRecQ1, a QDE-3 homologue in rice, is required for RNA silencing induced by particle bombardment for inverted repeat DNA, but not for double-stranded RNA.

Based on the nucleotide sequence of QDE-3 in Neurospora crassa, which is involved in RNA silencing, rice (Oryza sativa) mutant lines disrupted by the insertion of the rice retrotransposon Tos17 were selected. Homozygous individuals from the M(1) and M(2) generations were screened and used for further analyses. The expression of the gene was not detected in leaves or calli of the mutant lines, in contrast to the wild type (WT). Induction of RNA silencing by particle bombardment was performed to investigate any effects of the OsRecQ1 gene on RNA silencing with silencing inducers of the GFP (green fluorescence protein)/GUS (beta-glucuronidase) gene in the mutant lines. The results showed that OsRecQ1 is required for RNA silencing induced by particle bombardment for inverted-repeat DNA, but not for double-stranded RNA (dsRNA). The levels of transcripts from inverted-repeat DNA were much lower in the mutant lines than those in the WT. Furthermore, no effects were observed in the accumulation of endogenous microRNAs (miR171 and miR156) and the production of the short interspersed nuclear element retroelement by small interfering RNA. On the basis of these results, we propose that OsRecQ1 may participate in the process that allows inverted repeat DNA to be transcribed into dsRNA, which can trigger RNA silencing.

Restriction landmark genome scanning method using isoschizomers (MspI/HpaII) for DNA methylation analysis.

Restriction landmark genome scanning (RLGS) is a 2-DE of genomic DNA, which visualizes thousands of loci. In a conventional RLGS method for methylation analysis, we have used a methylation sensitive restriction enzyme, NotI as a landmark. However, it was unable to discriminate methylation polymorphism from sequence polymorphism. Here, we report an improved RLGS method to detect methylated sites directly. We employed isoschizomers, MspI and HpaII, that recognize the same sequence (CCGG) but have different methylation sensitivity. We carried out the RLGS analysis of Arabidopsis thaliana ecotype Columbia, and obtained a pair of spot patterns with MspI and HpaII. We detected 22 spots in both patterns. In comparison of them, 18% of the spots were polymorphic, which indicated the methylation of C(5m)CGG sites. Further analyses revealed an additional methylated site of NotI. Moreover, 52 and 54 restriction enzyme sites were also analyzed in two other ecotypes, Wassilewskija and Landsberg erecta, respectively. Consequently, 15% of the 52 common sites showed methylation polymorphism among the three ecotypes. The restriction sites analyzed in this study were located in or near genes, and contribute new data about the correlation between methylation status and gene expression. Therefore, this result strongly indicates that the improved RLGS method is readily applicable to practical analyses of methylation dynamics, and provides clues to the relationship between methylation and gene expression.