Fukashi Shibata

Chromosome painting of Y chromosomes and isolation of a Y chromosome-specific repetitive sequence in the dioecious plant Rumex acetosa.

Abstract.The dioecious plant Rumex acetosa has a multiple sex chromosome system: XX in female and XY1Y2 in male. Both types of Y chromosome were isolated from chromosome spreads of males by manual microdissection, and their chromosomal DNA was amplified using degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR). When the biotin-labeled DOP-PCR product was hybridized with competitor DNA in situ, the fluorescent signal painted the Y chromosomes. A library of Y chromosome DNA was constructed from the DOP-PCR product and screened for DNA sequences specific to the Y chromosome. One Y chromosome-specific DNA sequence was identified and designated RAYSI (R. acetosa Y chromosome-specific sequence I). RAYSI is a tandemly arranged repetitive DNA sequence that maps to the 4’,6-diamidino-2-phenylindole bands of both Y chromosomes.

Chromosome identification and comparative karyotypic analyses of four Pinus species

Abstract.Chromosomal landmarks in four Pinus species: P. densiflora, P. thunbergii, P. sylvestris, and P. nigra were identified by fluorescence in situ hybridization (FISH) using hapten- or fluorochrome-labeled probes for the plant telomere repeat, centromeric repeat (PCSR), and rDNA. FISH landmarks were located at the interstitial and proximal regions of chromosomes and allowed us to identify nearly all of the homologous chromosomes in each species. A comparative analysis of the FISH karyotypes among the four species showed that the interstitial FISH signals obtained by hybridization with the telomere and rDNA sequences were stable and could be used to identify homologous chromosomes among species. The identification of homologous chromosomes among species facilitated a detailed comparative karyotype analysis. The results suggest that the degree of chromosomal differentiation among the four Pinus species is very low and that the proximal regions vary in their DNA sequences. The similarities and differences among FISH karyotypes are discussed in relation to phylogeny.

Differential localization of the centromere-specific proteins in the major centromeric satellite of Arabidopsis thaliana

The 180 bp family of tandem repetitive sequences, which constitutes the major centromeric satellite in Arabidopsis thaliana, is thought to play important roles in kinetochore assembly. To assess the centromere activities of the 180 bp repeats, we performed indirect fluorescence immunolabeling with antibodies against phosphorylated histone H3 at Ser10, HTR12 (Arabidopsis centromeric histone H3 variant) and AtCENP-C (Arabidopsis CENP-C homologue) for the A. thaliana cell cultures. The immunosignals from all three antibodies appeared on all sites of the 180 bp repeats detected by fluorescence in situ hybridization. However, some of the 180 bp repeat clusters, particularly those that were long or stretched at interphase, were not fully covered with the signals from anti-HTR12 or AtCENP-C. Chromatin fiber immunolabeling clearly revealed that the centromeric proteins examined in this study, localize only at the knobs on the extended chromatin fibers, which form a limited part of the 180 bp clusters. Furthermore, outer HTR12 and inner phosphohistone H3 (Ser10) localization at the kinetochores of metaphase chromosomes suggests that two kinds of histone H3 (a centromere variant and a phosphorylated form) might be linked to different roles in centromere functionality; the former for spindle-fiber attachment, and the latter for chromatid cohesion.

Differentiation and the polymorphic nature of the Y chromosomes revealed by repetitive sequences in the dioecious plant, Rumex acetosa.

The dioecious plant Rumex acetosa has a multiple sex chromosome system: females are 2n = XX + 12, males are 2n = XY1Y2 + 12, and the two Y chromosomes are heterochromatic. A DNA sequence abounded in the male genome was isolated and analyzed. The sequence (RAE180) was a 180-bp-long tandemly arranged repetitive sequence, distributed in chromosomes Y1 and Y2, and two pairs of autosomes. Both Y chromosomes contained large amounts of RAE180 and the sequence formed many DAPI bands, while, on the two pairs of autosomes, RAE180 did not form DAPI bands. The internal structure and morphological changes of the Y chromosomes were analyzed by FISH, using RAE180 and the Y-chromosome-specific sequence RAYSI as probes. The pattern of the FISH signals caused by the accumulation of RAE180 and RAYSI suggested the structural change in the Y chromosomes during the process of sex chromosome evolution, and the morphological change in the Y chromosomes was explained by reciprocal translocation and inversion.

Longicorn beetle that vectors pinewood nematode carries many Wolbachia genes on an autosome

Monochamus alternatus is the longicorn beetle notorious as a vector of the pinewood nematode that causes the pine wilt disease. When two populations of M. alternatus were subjected to diagnostic polymerase chain reaction (PCR) detection of four Wolbachia genes, only the ftsZ gene was detected from one of the populations. The Wolbachia ftsZ gene persisted even after larvae were fed with a tetracycline-containing diet for six weeks. The inheritance of the ftsZ gene was not maternal but biparental, exhibiting a typical Mendelian pattern. The ftsZ gene titres in homozygotic ftsZ+ insects were nearly twice as high as those in heterozygotic ftsZ+ insects. Exhaustive PCR surveys revealed that 31 and 30 of 214 Wolbachia genes examined were detected from the two insect populations, respectively. Many of these Wolbachia genes contained stop codon(s) and/or frame shift(s). Fluorescent in situ hybridization confirmed the location of the Wolbachia genes on an autosome. On the basis of these results, we conclude that a large Wolbachia genomic region has been transferred to and located on an autosome of M. alternatus. The discovery of massive gene transfer from Wolbachia to M. alternatus would provide further insights into the evolution and fate of laterally transferred endosymbiont genes in multicellular host organisms.

Characterization of a Mis12 homologue in Arabidopsis thaliana

The centromere/kinetochore represents an important complex on chromosomes that contains a large number of proteins and facilitates accurate chromosome segregation during cell division. Fission yeast Mis12 and its human homologue hMis12 have been identified as essential kinetochore components. Although homologues have been suggested to exist in plants, their function remains to be determined. In this study the full-length cDNA of the Mis12 homologue from Arabidopsis thaliana (AtMIS12) was successfully cloned by RACE-and RT-PCR and the DNA sequence determined. The 238 amino acid sequence deduced from the cDNA contains two conserved blocks and a coiled-coil motif, despite the poor overall similarity to fission yeast and human Mis12. The antibody raised against a partial peptide of AtMIS12 recognized a 27-kDa protein corresponding to the predicted molecular weight. Immunofluorescence labeling using the antibody revealed that AtMIS12 localizes at centromeric regions, like the centromeric histone H3 variant HTR12, throughout the cell cycle. These results indicate that AtMIS12 is a constitutive component of Arabidopsis kinetochores.

Extensive Conserved Synteny of Genes Between the Karyotypes of Manduca sexta and Bombyx mori Revealed by BAC-FISH Mapping

Background Genome sequencing projects have been completed for several species representing four highly diverged holometabolous insect orders, Diptera, Hymenoptera, Coleoptera, and Lepidoptera. The striking evolutionary diversity of insects argues a need for efficient methods to apply genome information from such models to genetically uncharacterized species. Constructing conserved synteny maps plays a crucial role in this task. Here, we demonstrate the use of fluorescence in situ hybridization with bacterial artificial chromosome probes as a powerful tool for physical mapping of genes and comparative genome analysis in Lepidoptera, which have numerous and morphologically uniform holokinetic chromosomes. Methodology/Principal Findings We isolated 214 clones containing 159 orthologs of well conserved single-copy genes of a sequenced lepidopteran model, the silkworm, Bombyx mori, from a BAC library of a sphingid with an unexplored genome, the tobacco hornworm, Manduca sexta. We then constructed a BAC-FISH karyotype identifying all 28 chromosomes of M. sexta by mapping 124 loci using the corresponding BAC clones. BAC probes from three M. sexta chromosomes also generated clear signals on the corresponding chromosomes of the convolvulus hawk moth, Agrius convolvuli, which belongs to the same subfamily, Sphinginae, as M. sexta. Conclusions/Significance Comparison of the M. sexta BAC physical map with the linkage map and genome sequence of B. mori pointed to extensive conserved synteny including conserved gene order in most chromosomes. Only a few rearrangements, including three inversions, three translocations, and two fission/fusion events were estimated to have occurred after the divergence of Bombycidae and Sphingidae. These results add to accumulating evidence for the stability of lepidopteran genomes. Generating signals on A. convolvuli chromosomes using heterologous M. sexta probes demonstrated that BAC-FISH with orthologous sequences can be used for karyotyping a wide range of related and genetically uncharacterized species, significantly extending the ability to develop synteny maps for comparative and functional genomics.

Evolution of 5S rDNA units and their chromosomal localization in Allium cepa and Allium schoenoprasum revealed by microdissection and FISH

Abstract.Allium cepa and Allium schoenoprasum each possess 5S rDNA units of two different sizes. The evolution of the two repeat units and their chromosomal localization were investigated. A. cepa has 5S rDNA loci in the proximal and distal regions of the short arm of chromosome 7. When the proximal and distal segments of the short arm of chromosome 7 were microdissected separately, and used as templates for PCR, the short and long 5S rDNA fragments were amplified predominantly from the proximal and distal segments, respectively. The nucleotide sequence of the long 5S rDNA unit resulted from partial duplication of a non-transcribed spacer (NTS) and the insertion of a unique sequence. FISH using a probe consisting of the unique sequence demonstrated that the long unit was distally localized. In A. cepa, the long 5S rDNA unit is only present distally and the short unit is predominantly located proximally on the short arm of chromosome 7. In A. schoenoprasum, the NTSs of the two different-sized 5S rDNAs had quite different sequences. The two 5S rDNA loci were localized very close together in the interstitial region of chromosome 6. FISH, using long and short 5S rDNA unit probes with a competitor of a 120-bp sequence of the 5S rRNA gene, indicated that the long 5S rDNA unit was localized proximally and the short unit distally. Although the NTSs of the 5S rDNA of A. cepa and A. schoenoprasum had quite different nucleotide sequences, the long 5S rDNA units of A. cepa and A. schoenoprasum share a common 75-bp sequence. This sequence might act in the formation of the long 5S rDNA unit in Allium species.

The identification and analysis of the sequences that allow the detection of Allium cepa chromosomes by GISH in the allodiploid A. wakegi

Abstract. In Allium wakegi, which is an allodiploid species between Allium cepa and Allium fistulosum, each genome can be clearly distinguished using genomic in situ hybridization (GISH). Genomic DNA of A. cepa and A. fistulosum is differentiated both qualitatively and quantitatively. We wanted to isolate nucleotide sequences that give genome-specific signals on A. cepa chromosomes in GISH experiments in A. wakegi. We isolated 23 clones that show GISH-like signal patterns in fluorescence in situ hybridization (FISH) and analyzed their distribution in the A. cepa- and A. fistulosum-derived genomes of A. wakegi. There was considerable variation in the abundance and distribution of these cloned sequences on the chromosomes of the two species. The degree of A. cepa specificity varied among the clones. Twenty-two of the clones showed an even distribution over most chromosome arms with some clustering in the pericentromeric regions, but one clone showed very distinct terminal signals on some chromosomes. Whereas these sequences are not specific for A. cepa, changes in bases in nucleotide sequences and in their amount result in genome-specific characteristics in GISH experiments.

Functional analysis of the Arabidopsis centromere by T-DNA insertion-induced centromere breakage.

Two minichromosomes (α and δ) in addition to two other aberrant chromosomes (β and γ) were found in a transgenic Arabidopsis plant produced by an in planta vacuum infiltration technique. The minichromosomes were successfully separated by successive crossing and selfing and added to wild-type Columbia (Col-0) as a supernumerary chromosome. FISH indicated that both of the two minichromosomes originated from the short arm of chromosome 2. The mini α chromosome contained the whole short-arm 2S and a truncated centromere (180-bp repeat cluster), whereas mini δ lacked the terminal region including telomere repeats. Pachytene FISH clearly revealed that mini δ comprised a ring chromosome carrying two copies of the region from the 180-bp repeat cluster to BAC-F3C11. Both of the 180-bp clusters (each ≈500 kb in length) were thought to possess normal centromere functions because the centromere-specific histone H3 variant (HTR12) was detected on both clusters. Notwithstanding this dicentric and ring form, mini δ was stably transmitted to the next generations, perhaps because of its compact size (<4 Mb). Chromosome β also comprised a dicentric-like structure, with one of the two 180-bp repeat sites derived from chromosome 1 and the other from chromosome 2. However, the latter was quite small and failed to bind HTR12. The data obtained in this study indicated that 500 kb of the 180-bp array of the chromosome 2 centromere, from the edge of the 180-bp array on the short-arm side, is sufficient to form a functional domain.