Hisashi Tsujimoto

De novo synthesis of telomere sequences at the healed breakpoints of wheat deletion chromosomes.

Abstract When chromosomes are broken, the breakpoints become highly unstable and acquire the ability to fuse with other broken ends. The breakpoints are, however, eventually stabilized, and, therefore, the broken chromosomes are transmitted to the daughter cells without further morphological change. This phenomenon, known as “healing of breakpoints”, involves the addition of repetitive telomere sequences at the breakpoints by telomerase, the enzyme that normally synthesizes the telomere sequence at normal chromosome terminals. In many higher organisms, however, this property has not been well investigated. In this study, we examined the telomere sequences in wheat deletion lines with breakpoints on chromosome 1B. Lines that had breakpoints around the nucleolar organizer region were first selected on the basis of cytological observations, and the precise breakpoints were determined by mapping a fragment of rDNA and RFLP markers. In three lines – in addition to one previously reported – the DNA fragments encompassing the breakpoints were amplified by PCR using primers located in the rDNA and in telomere sequences. The DNA sequences provide insight into the properties of the telomerase activity at the breakpoints. The telomere sequences initiated from 2- to 4-nucleotide motifs in the original ribosomal DNA sequence which are also found in the repeat unit characteristic of telomere sequences. No specific sequences or structures were observed at or around the breakpoints. At all of the four breakpoints investigated, the newly synthesized telomere sequences contained considerable numbers of atypical telomere sequence units, particularly TTAGGG, which is the common unit of mammalian telomere sequences. Based on these results, we discuss the ability of plant telomerase to initiate the de novo synthesis of telomere sequences at internal breakpoints.

Proteome approaches to characterize seed storage proteins related to ditelocentric chromosomes in common wheat (Triticum aestivum L.)

Changes in protein composition of wheat endosperm proteome were investigated in 39 ditelocentric chromosome lines of common wheat (Triticum aestivum L.) cv. Chinese Spring. Two‐dimensional gel electrophoresis followed by Coomassie Brilliant Blue staining has resolved a total of 105 protein spots in a gel. Quantitative image analysis of protein spots was performed by PDQuest. Variations in protein spots between the euploid and the 39 ditelocentric lines were evaluated by spot number, appearance, disappearance and intensity. A specific spot present in all gels was taken as an internal standard, and the intensity of all other spots was calculated as the ratio of the internal standard. Out of the 1755 major spots detected in 39 ditelocentric lines, 1372 (78%) spots were found variable in different spot parameters: 147 (11%) disappeared, 978 (71%) up‐regulated and 247 (18%) down‐regulated. Correlation studies in changes in protein intensities among 24 protein spots across the ditelocentric lines were performed. High correlations in changes of protein intensities were observed among the proteins encoded by genes located in the homoeologous arms. Locations of structural genes controlling 26 spots were identified in 10 chromosomal arms. Multiple regulators of the same protein located at various chromosomal arms were also noticed. Identification of structural genes for most of the proteins was found difficult due to multiple regulators encoding the same protein. Two novel subunits (1BZ, 1BDz), the structure of which are very similar to the high molecular weight glutenin subunit 12, were identified, and the chromosome arm locations of these subunits were assigned.

A tandem repetitive sequence located in the centromeric region of common wheat (Triticum aestivum) chromosomes.

Although TaiI-family sequences are present in the subtelomeric region of Leymus racemosus, it became apparent in the present study that such sequences are also present in the centromeric region of common wheat (Triticum aestivum). These sequences hybridized to all chromosomes with various degrees of signal strength. FISH using TaiI and Ty3/gypsy, a conservative sequence in cereal centromeres, revealed a complicated arrangement of both sequences in all wheat chromosomes at once. Unlike the Arabidopsis centromeres characterized by massive tandem arrays of 180-bp family with flanking paracentromeric retrotransposons in all chromosomes, wheat chromosomes showed various arrangement patterns of TaiI and Ty3/gypsy sequences depending on the chromosome; TaiI-family sequences were scattered in many wheat centromeres as isolated colonies instead of forming uninterrupted solid tandem arrays. This pattern may have resulted from retrotransposon insertion within pre-existing TaiI-tandem arrays or a two-step amplification mechanism of the TaiI family where each TaiI colony was amplified to form arrays independently after the insertion of TaiI-family sequences along the entire centromere. Although sequence analysis of centromeric TaiI repeats in wheat and subtelomeric TaiI repeats in L. racemosus showed variable and conservative regions between the two repeats, they did not show a distinctive difference phylogenically. The widespread presence of tandem repetitive sequences in the eucaryotic centromere suggests a significant role for them in centromeric formation.

Exclusive localization of tandem repetitive sequences in subtelomeric heterochromatin regions of Leymus racemosus (Poaceae, Triticeae)

Two kinds of tandem repetitive sequences were isolated from Leymus racemosus (Lam.) Tzvelev. One of them was classified in the 350-bp family originally isolated from Secale. The other was a novel repetitive sequence family, named ‘TaiI family’, which consisted of a repeat unit of 570 bp. Fluorescence in-situ hybridization of the chromosomes of L. racemosus indicated that both families were located in subtelomeric heterochromatin and that the 350-bp family and TaiI family occupied different heterochromatin regions. In addition, even homologous chromosomes did not show the same patterns of TaiI and 350-bp families. The combination of these two families of repetitive sequences, together with Afa-family sequences and rDNAs, helps to identify the ten homologous chromosome pairs of L. racemosus. From these data, we proposed a karyotype of L. racemosus and compared it with other karyotypes already reported.

High-resolution cytological mapping of the long arm of chromosome 5A in common wheat using a series of deletion lines induced by gametocidal (Gc) genes of Aegilops speltoides

Gametocidal (Gc) genes of Aegilops in the background of the wheat genome lead to breakage of wheat chromosomes. The Q gene of wheat was used as a marker to select 19 deletion lines for the long arm of chromosome 5A of common wheat, Triticum aestivum cv. Chinese Spring (CS). The extents of deleted segments were cytologically estimated by the C-banding technique. The DNAs of deletion lines were hybridized with 22 DNA probes recognizing sites on the long arm of the chromosome (5AL) to determine their physical order. Based on the breeding behavior of the deletion lines, the location of a novel gene (Pv, pollen viability) affecting the viability of the male gamete was deduced. The segment translocated from 4AL to 5AL in CS was cytologically estimated to represent 13% of the total length of 5AL. Although DNA markers were almost randomly distributed along the chromosome arm, DNA markers located around the centromere and C-banded regions were obtained only rarely. Some deletion lines were highly rearranged in chromosome structure due to the effect(s) of the Gc gene. Applications of Gc genes for manipulating wheat chromosomes are discussed.

Molecular cytological evidence for gradual telomere synthesis at the broken chromosome ends in wheat

Telomere formation of the normal and broken chromosomes of common wheat,Triticum aestivum, was investigated byin situ hybridization using the biotin-labeled probe of telomere repetitive sequences (pAtT4) ofArabidopsis thaliana with subsequent amplification by an antibody. After double and triple amplification, prominent signals appeared at all the telomeric regions of the normal chromosomes.Prominent signals also emerged at the broken ends of the telocentric and deletion chromosomes that had passed through more than one generation since the appearance. However, broken ends that had passed through only the stages of gametogenesis, fertilization, embryogenesis and root development did not show complete signals such as found in normal telomeres. These findings indicate that a certain time or stage is required for synthesis of the telomeric repetitive sequences with a complete length. Nevertheless, because the broken ends without complete telomere sequences were also healed, restoration of the normal complement of telomere sequences is not necessary for healing of broken ends.

Chromosome assignment of four photosynthesis-related genes and their variability in wheat species.

Copy numbers of four photosynthesis-related genes, PhyA, Ppc, RbcS and Lhcb1*1, in wheat genomes were estimated by slot-blot analysis, and these genes were assigned to the chromosome arms of common wheat by Southern hybridization of DNA from an aneuploid series of the cultivar Chinese Spring. The copy number of PhyA was estimated to be one locus per haploid genome, and this gene was assigned to chromosomes 4AL, 4BS and 4DS. The Ppc gene showed a low copy number of small multigenes, and was located on the short arm of homoeologous group 3 chromosomes and the long arm of chromosomes of homoeologous group 7. RbcS consisted of a multigene family, with approximately 100 copies in the common wheat genome, and was located on the short arm of group 2 chromosomes and the long arm of group 5 chromosomes. Lhcb1*1 also consisted of a multigene family with about 50 copies in common wheat. Only a limited number of restriction fragments (approximately 15%) were used to determine the locations of members of this family on the long arm of group 1 chromosomes owing to the multiplicity of DNA bands. The variability of hybridized bands with the four genes was less in polyploids, but was more in the case of multigene families. RFLP analysis of polyploid wheats and their presumed ancestors was carried out with probes of the oat PhyA gene, the maize Ppc gene, the wheat RbcS gene and the wheat Lhcb1*1 gene. The RFLP patterns of common wheat most closely resembled those of T. Dicoccum (Emmer wheat), T. urartu (A genome), Ae. speltoides (S genome) and Ae. squarrosa (D genome). Diversification of genes in the wheat complex appear to have occurred mainly at the diploid level. Based on RFLP patterns, B and S genomes were clustered into two major groups. The fragment numbers per genome were reduced in proportion to the increase of ploidy level for all four genes, suggesting that some mechanism(s) might operate to restrict, and so keep to a minimum, the gene numbers in the polyploid genomes. However, the RbcS genes, located on 2BS, were more conserved (double dosage), indicating that the above mechanism(s) does not operate equally on individual genes.

A novel repetitive sequence, termed the JNK repeat family, located on an extra heterochromatic region of chromosome 2R of Japanese rye

Among cultivated rye, Secale cereale L., collected in Japan, we found an extra heterochromatin on the long-arm interstitial region of chromosome 2R. This extra heterochromatin was polymorphic in the population. The plants with the extra heterochromatin showed a specific DNA fragment of 1.2 kb in digests prepared with the restriction enzyme DraI. The fragment was cloned and used as a probe for fluorescent in-situ hybridization (FISH). The clone, pScJNK1, showed a hybridization signal at the extra heterochromatic region. The segregation of the number of signals corresponded to the number of the extra heterochromatin of the 2R chromosome, indicating that the sequence might construct the heterochromatin. Southern hybridization using the clone as a probe showed a ladder pattern, suggesting that the sequence was a tandem repeat. Three sequences homologous to pScJNK1 were isolated; these were 1192–1232 bp, 44.7–45.9% in GC content, highly homologous (>93%) with each other, and did not show any significant homology to other sequences in a DNA database. Slot blot hybridization using pScJNK1 as a probe indicated that there were about 4000 copies of the sequence in the haploid genome carrying the extra heterochromatin, whereas less than 20 copies existed in the genome without the heterochromatin. Southern hybridization using MspI and HapII indicated that all of the second cytosine nucleotides in CCGG sites in the sequence were methylated in the extra heterochromatin.

Identification of RFLP markers linked with heading date and its heterosis in hexaploid wheat

Recombinant inbred lines (RILs) from a cross between hexaploid wheat (T. aestivum cv. Chinese Spring (CS) and T. spelta (Sp)) were used for RFLP analysis of heading date and heterosis. Fourteen RFLP markers linking with heading date were identified; two were localized on chromosome 1A, one on 2A, three on 2B, one on 2D, four on 5A, two on 7A and one unlinked but reported to be on group 2. All of these markers may be attributable to genes for earliness per se. However, the markers in the chromosomes of 1A and 7A are new to this study. RILs were crossed with (tim)-CS, the alloplasmic CS with T. timopheevi cytoplasm, and the heterosis from earlier-parent and mid-parents were calculated for the F1s to examine the heterotic effect toward earliness on heading date. Five and two RFLP markers were associated with heterosis from the earlier-parent and mid-parents, respectively. They were distributed on the chromosomes of homoeologous groups 1 and 2.

Tetrad-FISH analysis reveals recombination suppression by interstitial heterochromatin sequences in rye (Secale cereale)

Abstract. Tetrad analysis is a genetic method that can locate genes and centromeres on a linkage map with a high degree of precision. Despite its effectiveness and accuracy, application of this method is generally limited to fungi, algae and mosses. Here we demonstrate a new method of tetrad analysis that is applicable to other organisms. This combines tetrad analysis with fluorescence in situ hybridization (FISH), and is thus referred to as tetrad-FISH analysis. We demonstrate the effectiveness of this method using tetrads of rye, Secale cereale. The rye strain JNK contains interstitial heterochromatin in a region of Chromosome 2R. We have previously cloned the tandemly repeated sequence forming this heterochromatin in the plasmid pScJNK. We performed FISH using pScJNK as the probe on tetrads obtained from heterozygotes for the heterochromatin region. The frequency of tetrads demonstrating positive signals in two cells that are diagonally opposite one another must correspond to the frequency of recombination in the interval between the heterochromatin and the centromere. Comparison between the results of tetrad-FISH analysis and linkage maps based on RFLP markers clearly indicated that heterochromatin strongly suppresses recombination of whole chromosomal regions. We discuss the effectiveness of tetrad-FISH analysis, particularly for the localization of functional centromeres in linkage maps.