Biomedical subjects

Shuhei Nasuda

Publications and source records attributed to Shuhei Nasuda.

12 recordsLinked to original sources

PubMed2006

Duplication of centromeric histone H3 (HTR12) gene in Arabidopsis halleri and A. lyrata, plant species with multiple centromeric satellite sequences.

Arabidopsis halleri and lyrata have three different major centromeric satellite sequences, a unique finding for a diploid Arabidopsis species. Since centromeric histones coevolve with centromeric satellites, these proteins would be predicted to show signs of selection when new centromere satellites have recently arisen. We isolated centromeric protein genes from A. halleri and lyrata and found that one of them, HTR12 (CENP-A), is duplicated, while CENP-C is not. Phylogenetic analysis indicates that the HTR12 duplication occurred after these species diverged from A. thaliana. Genetic mapping shows that HTR12 copy B has the same genomic location as the A. thaliana gene; the other copy (A, at the other end of the same chromosome) is probably the new copy. To test for selection since the duplication, we surveyed diversity at both HTR12 loci within A. lyrata. Overall, there is no strong evidence for an "evolutionary arms race" causing multiple replacement substitutions. The A. lyrata HTR12B sequences fall into three classes of haplotypes, apparently maintained for a long time, but they all encode the same amino acid sequence. In contrast, HTR12A has low diversity, but many variants are amino acid replacements, possibly due to independent selective sweeps within populations of the species.

Arabidopsis↗

PubMed2006

Polymorphic chromosomal specificity of centromere satellite families in Arabidopsis halleri ssp. gemmifera.

The chromosomal localizations of repetitive DNA clusters (ribosomal DNA and centromere satellites) were analyzed by fluorescent in situ hybridization in five strains of Arabidopsis halleri ssp. gemmifera. All five A. gemmifera strains have three chromosome pairs with 45S (5.8S-16S-26S) rDNA loci, and one pair with both 5S and 45S rDNA loci. These localizations are different from that of A. thaliana. Very unusually, there are three families of centromeric satellite DNAs (pAa, pAge1, and pAge2), and they showed polymorphism among the five strains studied. Overall, we found four different centromere satellite compositions. A plant from Fumuro was heterozygous for the chromosome specificities of centromere satellite families, possibly due to a reciprocal translocation involving centromere regions. Changes of centromeric satellite repeats appear to be rapid and frequent events in the history of A. gemmifera, and seem to occur by exchanging clusters as units.

Arabidopsis↗

PubMed2006

Chromosome-specific satellite sequences in Turritis glabra.

Two novel repetitive sequence families were isolated from Turritis glabra (2n = 2x = 12). These two repeat families are similar to those of centromeric repeats in Arabidopsis thaliana, are co-localized on one chromosome pair, and differ by about 20% from each other. Phylogenetic analysis revealed that the two repeat families of T. glabra are more similar to each other than to the centromeric repeat families of other Arabidopsis and related species. The relationships of satellite sequences reflected the species phylogeny, indicating that the replacement of satellite sequences has occurred in each species lineage independently, and shared variants could not have existed for a long time between species.

Arabidopsis↗

PubMed2005

Structural dynamics of cereal mitochondrial genomes as revealed by complete nucleotide sequencing of the wheat mitochondrial genome.

The application of a new gene-based strategy for sequencing the wheat mitochondrial genome shows its structure to be a 452 528 bp circular molecule, and provides nucleotide-level evidence of intra-molecular recombination. Single, reciprocal and double recombinant products, and the nucleotide sequences of the repeats that mediate their formation have been identified. The genome has 55 genes with exons, including 35 protein-coding, 3 rRNA and 17 tRNA genes. Nucleotide sequences of seven wheat genes have been determined here for the first time. Nine genes have an exon-intron structure. Gene amplification responsible for the production of multicopy mitochondrial genes, in general, is species-specific, suggesting the recent origin of these genes. About 16, 17, 15, 3.0 and 0.2% of wheat mitochondrial DNA (mtDNA) may be of genic (including introns), open reading frame, repetitive sequence, chloroplast and retro-element origin, respectively. The gene order of the wheat mitochondrial gene map shows little synteny to the rice and maize maps, indicative that thorough gene shuffling occurred during speciation. Almost all unique mtDNA sequences of wheat, as compared with rice and maize mtDNAs, are redundant DNA. Features of the gene-based strategy are discussed, and a mechanistic model of mitochondrial gene amplification is proposed.

Base Sequence↗

PubMed2005

An alternative to radiation hybrid mapping for large-scale genome analysis in barley.

The presence of a monosomic gametocidal chromosome (GC) in a barley chromosome addition line of common wheat generates structural aberrations in the barley chromosome as well as in the wheat chromosomes of gametes lacking the GC. A collection of structurally aberrant barley chromosomes is analogous to a panel of radiation hybrid (RH) mapping and is valuable for high-throughput physical mapping. We developed 90 common wheat lines (GC lines) containing aberrant barley 7H chromosomes induced by a gametocidal chromosome, 2C. DNAs isolated from these GC lines provided a panel of 7H chromosomal fragments in a wheat genetic background, comparable with RH mapping panels in mammals. We used this 7H GC panel and the methodology for RH mapping to physically map PCR-based barley markers, SSRs and AFLPs, onto chromosome 7H, relying on polymorphism between the 7H chromosome and the wheat genome. We call this method GC mapping. This study describes a novel adaptation and combination of methods of inducing chromosomal rearrangements to produce physical maps of markers. The advantages of the presented method are similar to RH mapping in that non-polymorphic markers can be used and the mapping panels can be relatively easily obtained. In addition, mapping results are cumulative when using the same mapping set with new markers. The GC lines will be available from the National Bioresources Project-KOMUGI ( http://www.nbrp.jp/index.jsp ).

Chromosomes, Plant↗

PubMed2005

Chromosomal assignment and deletion mapping of barley EST markers.

From about 10000 PCR-based EST markers of barley we chose 1421 EST markers that were demonstrated to be amplified differently by PCR between wheat (Triticum aestivum cv. Chinese Spring) and barley (Hordeum vulgare cv. Betzes). We assigned them to the seven barley chromosomes (1H to 7H) by PCR analysis using a set of wheat-barley chromosome addition lines. We successfully assigned 701 (49.3%) EST markers to the barley chromosomes: 75 to 1H, 127 to 2H, 119 to 3H, 94 to 4H, 108 to 5H, 81 to 6H and 97 to 7H. By using a set of Betzes barley telosomic addition lines of Chinese Spring, we could successfully determine the chromosome-arm (S or L) location of at least 90% of the EST markers assigned to each barley chromosome. We conducted a trial mapping using 90 EST markers assigned to 7HS (49) or 7HL (41) and 19 wheat lines carrying 7H structural changes. More EST markers were found in the distal region than in the proximal region.

Chromosome Deletion↗

PubMed2004

Durum wheat as a candidate for the unknown female progenitor of bread wheat: an empirical study with a highly fertile F1 hybrid with Aegilops tauschii Coss.

Hexaploid bread wheat was derived from a hybrid cross between a cultivated form of tetraploid Triticum wheat (female progenitor) and a wild diploid species, Aegilops tauschii Coss. (male progenitor). This cross produced a fertile triploid F1 hybrid that set hexaploid seeds. The identity of the female progenitor is unknown, but various cultivated tetraploid Triticum wheats exist today. Genetic and archaeological evidence suggests that durum wheat ( T. turgidum ssp. durum) may be the female progenitor. In previous studies, however, F1 hybrids of durum wheat crossed with Ae. tauschii consistently had low levels of fertility. To establish an empirical basis for the theory of durum wheat being the female progenitor of bread wheat, we crossed a durum wheat cultivar that carries a gene for meiotic restitution with a line of Ae. tauschii. F1 hybrids were produced without using embryo rescue techniques. These triploid F1 hybrids were highly fertile and spontaneously set hexaploid F2 seeds at the average selfed seedset rate of 51.5%. To the best of our knowledge, this is the first example of the production of highly fertile F1 hybrids between durum wheat and Ae. tauschii. The F1 and F2 hybrids are both similar morphologically to bread wheat and have vigorous growth habits. Cytological analyses of F1 male gametogenesis showed that meiotic restitution is responsible for the high fertility of the triploid F1 hybrids. The implications of these findings for the origin of bread wheat are discussed.

Crops, Agricultural↗

PubMed2004

Characterization of the genes encoding for MAD2 homologues in wheat.

MAD2 (mitotic arrest deficient 2) is a highly conserved protein involving in spindle checkpoint control. MAD2 locates at spindle-unattached kinetochores during prophase and dissolves from spindle-attached kinetochores towards metaphase. In this study, we isolated homologous genes encoding for MAD2 from hexaploid wheat. The three homoeologous genes on the long arms of the group-2 chromosomes shared approximately 99% similarity of the nucleotide sequence in coding regions. The intron-exon structures of the three homoeologues were also conserved, showing high similarities to that of the Arabidopsis MAD2 gene. All three homoeologues were transcribed in roots and spikes but not in leaves. We generated antibodies against the polypeptides with amino acid sequences derived from the cDNA sequences of the wheat MAD2 homologues. Using these antibodies, we found MAD2 in wheat root-tip cells to change in location and amount through the cell cycle, similar to those reported for human MAD2. Intense immunostaining signals were observed at the centromeres of all metaphase chromosomes when root-tips were treated with colchicine, a microtubule-destabilizing drug, but no signals were observed in untreated chromosomes. Thus, the wheat MAD2 protein could be a good marker for the functional kinetochores of metaphase chromosomes in wheat.

Amino Acid Sequence↗

PubMed2004

A direct repeat sequence associated with the centromeric retrotransposons in wheat.

A high-density BAC filter of Triticum monococcum was screened for the presence of a centromeric retrotransposon using the integrase region as a probe. Southern hybridization to the BAC digests using total genomic DNA probes of Triticum monococcum, Triticum aestivum, and Hordeum vulgare detected differentially hybridizing restriction fragments between wheat and barley. The fragments that hybridized to genomic DNA of wheat but not to that of barley were subcloned. Fluorescence in situ hybridization (FISH) analysis indicated that the clone pHind258 hybridized strongly to centromeric regions in wheat and rye and weakly to those in barley. The sequence of pHind258 was homologous to integrase and long terminal repeats of centromeric Ty3-gypsy retrotransposons of cereal species. Additionally, pHind258 has a pair of 192-bp direct repeats. FISH analysis indicated that the 192-bp repeat probe hybridized to centromeres of wheat and rye but not to those of barley. We found differential FISH signal intensities among wheat chromosomes using the 192-bp probe. In general, the A-genome chromosomes possess strong FISH signals, the B-genome chromosomes possess moderate signals, and the D-genome chromosomes possess weak signals. This was consistent with the estimated copy numbers of the 192-bp repeats in the ancestral species of hexaploid wheat.

Base Sequence↗

PubMed2004

A PCR-based marker for targeting small rye segments in wheat background.

We attempted to develop a PCR-based marker that detects various segments of rye chromosome incorporated into wheat. We designed three sets of PCR primers based on the nucleotide sequence data of a rye repetitive sequence previously reported. One of the primer sets amplified a clear ca. 1.4 kb fragment in a rye cultivar but not in any form of wheat, diploid, tetraploid or hexaploid. We used this critical primer set for PCR of various wild species and cultivars of rye, an array of wheat plants carrying different rye chromosomes or small segments from different regions of rye chromosome 1R, and plants carrying parts of the rye B chromosome. The PCR amplified the 1.4 kb fragment in all the plant materials examined. We believe this PCR primer set will be useful as a universal PCR-based marker for the introgression of rye chromosome segments in the wheat genome.

Chromosomes, Plant↗

PubMed2003

Characterization of a knock-out mutation at the Gc2 locus in wheat.

Gametocidal (Gc) genes, introduced into common wheat from related Aegilops species, are selfish genetic elements that ensure their preferential transmission by inducing chromosomal breaks. Here we report the production and characterization of a knock-out mutation of the Gc2 gene transferred to wheat as a wheat-Aegilops sharonensis T4B-4S(sh)#1 translocation chromosome. In hemizygous Gc2/- condition, gametophytes lacking Gc2 suffer chromosomal fragmentation and produce non-functional gametes, which leads to sporophytic semisterility and exclusive transmission of the Gc2-carrier chromosome. We have identified one putative ethyl methylsulfonate (EMS)-induced Gc2 mutant that restores spike fertility and shows Mendelian segregation. Progeny screening mapped the mutation to the Gc2-carrier chromosome T4B-4S(sh)#1. C-banding and fluorescence in situ hybridization analyses showed that the loss of Gc2 function in the mutant is not due to a terminal deficiency. Analysis of first and second pollen mitoses in Gc2(mut) /- plants and C-banding analysis of testcross progenies showed that no chromosomal breakage occurs in the mutant. No gametophytic chromosomal breakage was observed in heterozygous Gc2(mut) /Gc2 plants, which had fully fertile spikes. These results suggest that Gc2 encodes two agents, one causing chromosomal breaks in gametophytes lacking Gc2 and another that protects the Gc2 carrier from breakage. The EMS-induced Gc2 mutant appears to be a knock-out of the gene encoding the "breaking" agent. These data are a first crucial step toward the molecular understanding of Gc2 action.

Chromosome Breakage↗

PubMed2002

Molecular cloning, sequencing, and chromosome mapping of a 1A-encoded omega-type prolamin sequence from wheat.

Gliadins are the most abundant component of the seed storage proteins in cereals and, in combination with glutenins, are important for the bread-making quality of wheat. They are divided into four subfamilies, the alpha-, beta-, gamma-, and omega-gliadins, depending on their electrophoresis pattern, chromosomal location, and DNA and protein structures. Using a PCR-based strategy we isolated and sequenced an omega-gliadin sequence. We also determined the chromosomal subarm location of this sequence using wheat aneuploids and deletion lines. The gene is 1858 bp long and contains a coding sequence 1248 bp in length. Like all other gliadin gene families characterized in cereals, the omega-gliadin gene described here had characteristic features including two repeated sequences 300 bp upstream of the start codon. At the DNA level, the gene had a high degree of similarity to the omega-secalin and C-hordein genes of rye and barley, but exhibited much less homology to the alpha- and beta-gliadin gene families. In terms of the deduced amino acid sequence, this gene has about 80 and 70% similarity to the omega-secalin and C-hordein genes, respectively, and possesses all the features reported for other gliadin gene families. The omega-gliadin gene has about 30 repeats of the core consensus sequences PQQPX and XQQPQQX, twice as many as other gliadin gene families. Southern blotting and PCR analysis with aneuploid and deletion lines for the short arm of chromosome 1A showed that the omega-gliadin was located on the distal 25% of the short arm of chromosome 1A. By comparison of PCR and A-PAGE profiles for deletion stocks, its genomic location must be at a different locus from gli-Ala in 'Chinese Spring'.

Amino Acid Sequence↗