Hisato Okuizumi

Identification of an imprinted U2af binding protein related sequence on mouse chromosome 11 using the RLGS method

A new imprinted gene has been discovered in mice using the technique of restriction landmark genomic scanning (RGLS) with methylation sensitive enzymes. Eight out of 3,100 strain–specific Notl and BssHII spots were identified as imprinted in reciprocal F1 hybrids. Subsequently, we isolated a genomic clone for one locus on proximal chromosome 11 near the Glns locus, an imprinted region in uniparental disomic mice, and its corresponding cDNA clone. Expression of this transcript from the paternal allele was established using RT–PCR of reciprocal F1–hybrid mice. The amino–acid sequence deduced from the cDNA showed significant homology to the U2 small nuclear ribonucleoprotein auxiliary factor 35 kDa subunit.

Development of SSR-based sorghum (Sorghum bicolor (L.) Moench) diversity research set of germplasm and its evaluation by morphological traits

Assessment and utilization of diversity in plant genetic resources is vital for the improvement of plant species. A sorghum diversity research set (SDRS) was developed by using SSR markers. A total of 320 sorghum accessions were selected based on geographic distribution from more than 3,500 germplasm accessions comprising Asia (East, Southeast, South and Southwest Asia) and Africa, conserved at NIAS Genebank, Japan. We selected 38 simple sequence repeats (SSR) markers which generated 146 alleles, covering ten chromosomes of sorghum from a three different published SSR linkage map of sorghum. The average percentage of polymorphic loci (P) and gene diversity (He) observed in this study were 82.8 and 0.217 respectively. Analysis showed a positive correlation with geographic pattern of differentiation. Based on SSR assessment, 107 sorghum accessions were selected as diversity research set. There was no significant difference in pattern of genetic spectrum between SDRS and base population. Similarly no greater change was observed for variability parameters (Dice, %P, He) and almost all of the SSR alleles were retained in selected sorghum accessions except for the loss of a single allele at locus Xtxp287. SDRS was sown during sorghum sowing season in two replications. Data were recorded on 26 important morphological traits according to the standard sorghum descriptors at Genebank. Analysis of variance showed a highly significant difference among all accessions for all of the traits. Morpho-agronomic traits could not effectively classify the accessions according to geographic origin by using cluster analysis.

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

Abstract. 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.

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 x BIO 14.6) F1 x 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.

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”דKasalath”) 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.

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.

Protocols for RLGS Gel Production

RLGS can be performed using various enzyme combinations (restriction enzymes EL-EB-EC). The labeling method must be chosen according to the shape of the recognition site of enzyme EL. In addition, the reaction buffers used in each process should be changed, accompanied by variation of the enzyme. Here, protocols for two good representative enzyme combinations, combination 1 (NotI-PvuII-PstI) and combination 2 (PacI-EcoRV-MboI), are shown.

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 F1 hybrids (subsp. japonica cv. Nipponbare × 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 F1 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 F1 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 F1 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.

RLGS profile segmentation via a SVM

This paper presents a new and fast segmentation algorithm, capable of incorporating experts knowledge and expertise in a straightforward manner, to detect DNA spots in a restriction landmark genomic scanning (RLGS) profile. A RLGS profile is a gray scale image obtained in a X-ray film containing hundreds of dark spots corresponding to certain DNA fragments labeled by radioactive markers. Extracting these DNA spots is a very challenging task due to their fuzzy boundaries against noisy background regions having non-uniform intensity variations. The proposed algorithm, utilizing a support vector machine, is found to be successful to detect and extract DNA spots in a RLGS profile.

RLGS Spot Mapping Method

Recent progress in molecular genetics has been expedited by the development of DNA landmarks. The DNA landmarks visualized by Southern blot and PCR enabled us to construct high-density genome map which is essential for position-dependent identification of the gene responsible for a certain phenotype. This approach is so-called positional cloning. In the medical field, the information of the genes identified by position-dependent cloning (including positional candidate approach) is very useful for diagnosis, prevention, gene therapy, and drug therapy. This approach is the only known systematic way to connect any phenotypes to the transcripts, based on genetic analyses and molecular cloning technology. To facilitate the identification of genes using the positional cloning approach in higher organisms, high-density genetic maps which require a multiplex genome scanning method are essential. This is especially important in the case of orphan genomes which have important mutants but no dense map, so that we should employ a method that can rapidly scan a large number of loci.