Ultrafast Simultaneous Raman-Fluorescence Spectroscopy.

Raman and fluorescence spectroscopies offer complementary approaches in bioanalytical chemistry, particularly in microbiological assays. The former method is used to detect lipids, metabolites, and nonspecific proteins and nucleic acids in a label-free manner, while the latter is used to investigate targeted proteins, nucleic acids, and their interactions via labeling or transfection. Despite their complementarity, these regimes are seldom used in conjunction due to fluorescent signals overwhelming inherently weak Raman signals by more than several orders of magnitude. Here we report a multimodal spectrometer that simultaneously performs Raman and fluorescence spectroscopies at high speed. It is made possible by Fourier-transform-coherent anti-Stokes Raman scattering (FT-CARS) and Fourier-transform-two-photon excitation (FT-TPE) measurements powered by a femtosecond pulse laser coupled to a homemade rapid-scan Michelson interferometer, operating at 24 000 spectra per second. As a proof-of-principle demonstration, we validate the ultrafast fluoRaman spectrometer by measuring coumarin dyes in organic solvents. To show its potential for applications that require rapid fluoRaman spectroscopy, we also demonstrate fluoRaman flow cytometry of Haematococcus pluvialis cells under varying culture conditions with a high throughput of ∼10 events per second to perform large-scale single-cell analysis of their metabolic stress response.

Detection of Transgenes on DNA Fibers.

Fluorescence in situ hybridization (FISH) was developed for detecting specific DNA sequences directly on mitotic or meiotic chromosomes. However, the resolution of FISH on chromosomes is limited by condensed structure of chromatin, and it is difficult to differentiate two target sites close to each other. To overcome this issue, the objects was changed to stretched DNA fibers, and this fiber FISH technique has now been used for revealing genome structure at molecular level. Hybridization and detection procedures of fiber FISH are common with FISH on chromosomes. Therefore, application of fiber FISH is not difficult for the researchers of some experience in ordinary FISH. DNA fibers can be released from nuclei fixed on glass slides using a detergent. The DNA fibers were shred in FISH procedure, and the resultant fragments became small bead-like shape. This makes FISH signals on DNA fibers a series of dots. The size of DNA in the dot is estimated to be approximately 1 kb, it corresponding to the resolution of fiber FISH. This makes it possible to analyze structures of transgenes on DNA fibers in detail.

Rapid degeneration of noncoding DNA regions surrounding SlAP3X/Y after recombination suppression in the dioecious plant Silene latifolia.

Silene latifolia is a dioecious plant with heteromorphic XY sex chromosomes. Previous studies of sex chromosome-linked genes have suggested a gradual divergence between the X-linked and the Y-linked genes in proportion to the distance from the pseudoautosomal region. However, such a comparison has yet to be made for the noncoding regions. To better characterize the nonrecombining region of the X and Y chromosomes, we sequenced bacterial artificial chromosome clones containing the sex chromosome-linked paralogs SlAP3X and SlAP3Y, including 115 kb and 73 kb of sequences, respectively, flanking these genes. The synonymous nucleotide divergence between SlAP3X and SlAP3Y indicated that recombination stopped approximately 3.4 million years ago. Sequence homology analysis revealed the presence of six long terminal repeat retrotransposon-like elements. Using the nucleotide divergence calculated between left and right long terminal repeat sequences, insertion dates were estimated to be 0.083-1.6 million years ago, implying that all elements detected were inserted after recombination stopped. A reciprocal sequence homology search facilitated the identification of four homologous noncoding DNA regions between the X and Y chromosomes, spanning 6.7% and 10.6% of the X chromosome-derived and Y chromosome-derived sequences, respectively, investigated. Genomic Southern blotting and fluorescence in situ hybridization showed that the noncoding DNA flanking SlAP3X/Y has homology to many regions throughout the genome, regardless of whether they were homologous between the X and Y chromosomes. This finding suggests that most noncoding DNA regions rapidly lose their counterparts because of the introduction of transposable elements and indels (insertion-deletions) after recombination has stopped.

Tobacco karyotyping by accurate centromere identification and novel repetitive DNA localization.

Tobacco (Nicotiana tabacum) is an amphidiploid species (2n = 4x = 48, genome constitution SSTT) derived from a natural hybrid between Nicotiana sylvestris (2n = 2x = 24, SS) and Nicotiana tomentosiformis (2n = 2x = 24, TT). Genomic in situ hybridization (GISH), using the genomic DNA from these ancestral species as probes, revealed the chromosomal origins (S or T) and the occurrence of intergenomic translocations in N. tabacum. Fluorescence in situ hybridization (FISH) was also used to distinguish between chromosomes. However, the use of repetitive DNA sequences as probes for FISH analysis is limited by an inability to identify all chromosomes. In addition to this limitation, the occurrence of chromosomal tertiary constrictions can easily lead to the misclassification of chromosomes. To overcome these issues, immunostaining with anti-N. tabacum centromere-specific histone H3 antibody was carried out to determine the centromere position of each chromosome, followed by FISH analysis with ten distinct repetitive DNA probes. This approach allowed us to identify 22 of the 24 chromosome pairs in N. tabacum and revealed novel intergenomic chromosome rearrangements and B-chromosome-like minichromosomes. Hence, the combination of immunostaining with FISH and GISH is critical to accurately karyotype tobacco.

FISH identification of Helicoverpa armigera and Mamestra brassicae chromosomes by BAC and fosmid probes.

Since the Bombyx mori genome sequence was published, conserved synteny between B. mori and some other lepidopteran species has been revealed by either FISH (fluorescence in situ hybridization) with BAC (bacterial artificial chromosome) probes or linkage analysis. However, no species belonging to the Noctuidae, the largest lepidopteran family which includes serious polyphagous pests, has been analyzed so far with respect to genome-wide conserved synteny and gene order. For that purpose, we selected the noctuid species Helicoverpa armigera and Mamestra brassicae, both with n = 31 chromosomes. Gene-defined fosmid clones from M. brassicae and BAC clones from a closely related species of H. armigera, Heliothis virescens, were used for a FISH analysis on pachytene chromosomes. We recognized all H. armigera chromosomes from specific cross-hybridization signals of 146 BAC probes. With 100 fosmid clones we identified and characterized all 31 bivalents of M. brassicae. Synteny and gene order were well conserved between the two noctuid species. The comparison with the model species B. mori (n = 28) showed the same phenomenon for 25 of the 28 chromosomes. Three chromosomes (#11, #23 and #24) had two counterparts each in H. armigera and M. brassicae. Since n = 31 is the modal chromosome number in Lepidoptera, the noctuid chromosomes probably represent an ancestral genome organization of Lepidoptera. This is the first identification of a full karyotype in Lepidoptera by means of BAC cross-hybridization between species. The technique shows the potential to expand the range of analyzed species efficiently.

Generation of an artificial ring chromosome in Arabidopsis by Cre/LoxP-mediated recombination.

A eukaryotic chromosome consists of a centromere, two telomeres and a number of replication origins, and 'artificial chromosomes' may be created in yeast and mammals when these three elements are artificially joined and introduced into cells. Plant artificial chromosomes (PACs) have been suggested as new vectors for the development of new crops and as tools for basic research on chromosomes. However, indisputable PAC formation has not yet been confirmed. Here, we present a method for generating PACs in the model plant Arabidopsis thaliana using the Cre/LoxP and Activator/Dissociation element systems. The successfully generated PAC, designated AtARC1 (A. thaliana artificial ring chromosome 1), originated from a centromeric edge of the long arm of chromosome 2, but its size (2.85 Mb) is much smaller than that of the original chromosome (26.3 Mb). Although AtARC1 contains only a short centromere domain consisting of 180 bp repeats approximately 250 kb in length, compared with the 3 Mb domain on the original chromosome 2, centromere-specific histone H3 (HTR12) was detected on the centromeric region. This result supported the observed stability of the PAC during mitosis in the absence of selection, and transmission of the PAC to the next generation through meiosis. Because AtARC1 contains a unique LoxP site driven by the CaMV 35S promoter, it is possible to introduce a selectable marker and desired transgenes into AtARC1 at the LoxP site using Cre recombinase. Therefore, AtARC1 meets the criteria for a PAC and is a promising vector.

EST sequencing and fosmid library construction in a non-model moth, Mamestra brassicae, for comparative mapping.

Genome data are useful for both basic and applied research; however, it is difficult to carry out large-scale genome analyses using species with limited genetic or genomic resources. Here, we describe a cost-effective method to analyze the genome of a non-model species, using the cabbage moth, Mamestra brassicae (Lepidoptera: Noctuidae). First, we conducted expression sequence tag (EST) analysis. In this analysis, we performed PCR-based prescreening of a non-normalized embryonic cDNA library to eliminate already sequenced cDNAs from further sequencing, which significantly increased the percentage of unique genes. Next, we constructed a fosmid library of M. brassicae and isolated 120 clones containing 119 putative single copy genes by PCR-based screening with primer sets designed from the ESTs. Finally, we showed that the isolated fosmid clones could be used as probes for multicolor fluorescence in situ hybridization (FISH) analysis against an M. brassicae chromosome and confirmed conserved gene order between M. brassicae and the silkworm, Bombyx mori. Thus, we developed new genomic resources for comparative genome analysis in M. brassicae using robust and relatively low cost methods that can be applied to any non-model organism.

Isolation of centromeric-tandem repetitive DNA sequences by chromatin affinity purification using a HaloTag7-fused centromere-specific histone H3 in tobacco.

The centromere is a multi-functional complex comprising centromeric DNA and a number of proteins. To isolate unidentified centromeric DNA sequences, centromere-specific histone H3 variants (CENH3) and chromatin immunoprecipitation (ChIP) have been utilized in some plant species. However, anti-CENH3 antibody for ChIP must be raised in each species because of its species specificity. Production of the antibodies is time-consuming and costly, and it is not easy to produce ChIP-grade antibodies. In this study, we applied a HaloTag7-based chromatin affinity purification system to isolate centromeric DNA sequences in tobacco. This system required no specific antibody, and made it possible to apply a highly stringent wash to remove contaminated DNA. As a result, we succeeded in isolating five tandem repetitive DNA sequences in addition to the centromeric retrotransposons that were previously identified by ChIP. Three of the tandem repeats were centromere-specific sequences located on different chromosomes. These results confirm the validity of the HaloTag7-based chromatin affinity purification system as an alternative method to ChIP for isolating unknown centromeric DNA sequences. The discovery of more than two chromosome-specific centromeric DNA sequences indicates the mosaic structure of tobacco centromeres.

Stability of monocentric and dicentric ring minichromosomes in Arabidopsis.

A dicentric ring minichromosome (miniδ) was identified in transgenic Arabidopsis thaliana and added to a wild type as a supernumerary chromosome. This line is relatively stable and has been maintained for generations, notwithstanding its ring and dicentric structure. To determine the mechanism for stable transmission of miniδ, the structure and behavior of two new types of ring minichromosomes (miniδ1 and miniδ1-1) derived from miniδ were investigated. Fluorescence in situ hybridization analysis revealed that miniδ1 is dicentric just like miniδ, whereas miniδ1-1 is monocentric. The estimated sizes of miniδ1 and miniδ1-1 were 3.8~5.0 and 1.7 Mb, respectively. The sizes of the two centromeres on miniδ1 were identical (ca. 270 kb) and similar to that of miniδ1-1 (ca. 250 kb). Miniδ1 was relatively stable during mitosis and meiosis, as is miniδ, whereas miniδ1-1 was unstable during mitosis, and the number of minichromosomes per cell varied. This possibly resulted from misdivision caused by a short centromere on monocentric miniδ1-1. Transmission through the female was quite limited for all three ring minichromosomes (0-3.2%), whereas that through the male was relatively high (15.4-27.3%) compared with that of other supernumerary chromosomes in Arabidopsis. Ring structure without telomeres itself seems not to limit the female transmission.

Meiosis-specific loading of the centromere-specific histone CENH3 in Arabidopsis thaliana.

Centromere behavior is specialized in meiosis I, so that sister chromatids of homologous chromosomes are pulled toward the same side of the spindle (through kinetochore mono-orientation) and chromosome number is reduced. Factors required for mono-orientation have been identified in yeast. However, comparatively little is known about how meiotic centromere behavior is specialized in animals and plants that typically have large tandem repeat centromeres. Kinetochores are nucleated by the centromere-specific histone CENH3. Unlike conventional histone H3s, CENH3 is rapidly evolving, particularly in its N-terminal tail domain. Here we describe chimeric variants of CENH3 with alterations in the N-terminal tail that are specifically defective in meiosis. Arabidopsis thaliana cenh3 mutants expressing a GFP-tagged chimeric protein containing the H3 N-terminal tail and the CENH3 C-terminus (termed GFP-tailswap) are sterile because of random meiotic chromosome segregation. These defects result from the specific depletion of GFP-tailswap protein from meiotic kinetochores, which contrasts with its normal localization in mitotic cells. Loss of the GFP-tailswap CENH3 variant in meiosis affects recruitment of the essential kinetochore protein MIS12. Our findings suggest that CENH3 loading dynamics might be regulated differently in mitosis and meiosis. As further support for our hypothesis, we show that GFP-tailswap protein is recruited back to centromeres in a subset of pollen grains in GFP-tailswap once they resume haploid mitosis. Meiotic recruitment of the GFP-tailswap CENH3 variant is not restored by removal of the meiosis-specific cohesin subunit REC8. Our results reveal the existence of a specialized loading pathway for CENH3 during meiosis that is likely to involve the hypervariable N-terminal tail. Meiosis-specific CENH3 dynamics may play a role in modulating meiotic centromere behavior.

Coexistence of NtCENH3 and two retrotransposons in tobacco centromeres.

Although a centromeric DNA fragment of tobacco (Nicotiana tabacum), Nt2-7, has been reported, the overall structure of the centromeres remains unknown. To characterize the centromeric DNA sequences, we conducted a chromatin immunoprecipitation assay using anti-NtCENH3 antibody and chromatins isolated from two ancestral diploid species (Nicotiana sylvestris and Nicotiana tomentosiformis) of N. tabacum and isolated a 178-pb fragment, Nto1 from N. tomentosiformis, as a novel centromeric DNA. Fluorescence in situ hybridization (FISH) showed that Nto1 localizes on 24 out of 48 chromosomes in some cells of a BY-2 cell line. To identify the origins of the Nt2-7 and Nto1, a tobacco bacterial artificial chromosome (BAC) library was constructed from N. tabacum, and then screened by polymerase chain reaction (PCR) with primer sets designed from the Nt2-7 and Not1 DNA sequences. Twelve BAC clones were found to localize on the centromeric regions by FISH. We selected three BAC clones for sequencing and identified two centromeric retrotransposons, NtCR and NtoCR, the DNA sequences of which are similar to that of Nt2-7 and Nto1, respectively. Quantitative PCR analysis using coprecipitated DNA with anti-NtCENH3 clearly showed coexistence of NtCENH3 with both retrotransposons. These results indicate the possibility that these two retrotransposons act as centromeric DNA sequences in tobacco. NtoCR was found to be specific to N. tomentosiformis and T genome of N. tabacum, and a NtCR-like centromeric retrotransposon (TGRIV) exists in tomato. This specificity suggests that the times of amplification of these centromeric retrotransposons were different.

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.

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.

Reprobing multicolor FISH preparations in lepidopteran chromosome.

Multicolor fluorescence in-situ hybridization (FISH) and subsequent reprobing of chromosome preparations increase the number of chromosomes and/or anchor loci on the chromosomes simultaneously identified. Reprobing techniques have been widely applied to chromosomes of vertebrates and plants. We have developed a novel reprobing protocol that utilizes multicolor FISH and bacterial artificial chromosome (BAC) probes to examine chromosome preparations in a model lepidopteran species, the silkworm, Bombyx mori. With standard two-color BAC-FISH, routinely used to map genes on B. mori chromosomes, we could localize only two probes on one preparation, whereas our new protocol combining five-color BAC-FISH and preparation reprobing enabled us to simultaneously map 10 probes, as demonstrated with the Bombyx Z chromosome. The improved BAC-FISH technique will facilitate karyotyping and synteny analysis in Lepidoptera.

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

Two minichromosomes (alpha and delta) in addition to two other aberrant chromosomes (beta and gamma) 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 alpha chromosome contained the whole short-arm 2S and a truncated centromere (180-bp repeat cluster), whereas mini delta lacked the terminal region including telomere repeats. Pachytene FISH clearly revealed that mini delta 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 approximately 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 delta was stably transmitted to the next generations, perhaps because of its compact size (<4 Mb). Chromosome beta 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.

Wolbachia genome integrated in an insect chromosome: evolution and fate of laterally transferred endosymbiont genes.

Recent accumulation of microbial genome data has demonstrated that lateral gene transfers constitute an important and universal evolutionary process in prokaryotes, while those in multicellular eukaryotes are still regarded as unusual, except for endosymbiotic gene transfers from mitochondria and plastids. Here we thoroughly investigated the bacterial genes derived from a Wolbachia endosymbiont on the nuclear genome of the beetle Callosobruchus chinensis. Exhaustive PCR detection and Southern blot analysis suggested that approximately 30% of Wolbachia genes, in terms of the gene repertoire of wMel, are present on the insect nuclear genome. Fluorescent in situ hybridization located the transferred genes on the proximal region of the basal short arm of the X chromosome. Molecular evolutionary and other lines of evidence indicated that the transferred genes are probably derived from a single lateral transfer event. The transferred genes were, for the length examined, structurally disrupted, freed from functional constraints, and transcriptionally inactive. Hence, most, if not all, of the transferred genes have been pseudogenized. Notwithstanding this, the transferred genes were ubiquitously detected from Japanese and Taiwanese populations of C. chinensis, while the number of the transferred genes detected differed between the populations. The transferred genes were not detected from congenic beetle species, indicating that the transfer event occurred after speciation of C. chinensis, which was estimated to be one or several million years ago. These features of the laterally transferred endosymbiont genes are compared with the evolutionary patterns of mitochondrial and plastid genome fragments acquired by nuclear genomes through recent endosymbiotic gene transfers.

The origin, meiotic behavior, and transmission of a novel minichromosome in Arabidopsis thaliana.

A plant carrying a small extra chromosome was found in Landsberg erecta ecotype of Arabidopsis thaliana. Fluorescence in situ hybridization revealed that this minichromosome was derived from the short arm of chromosome 4. The size of this "mini4S" chromosome was estimated to be approximately 7.5 Mb on the basis of previously reported data and the amount of the centromeric major satellite (180-bp family) present, which was determined to be about 1 Mb, or about one third of that in the normal chromosome 4. No pairing between mini4S and its original chromosome 4 was observed at pachytene and metaphase I stages. The transmission of mini4S through pollen was limited, but about 30% of selfed progeny carried the mini4S chromosomes. The transmission rates considerably increased when the mini4S chromosomes were transferred to plants with a Columbia background by successive backcrosses. This suggests that the stability of the minichromosomes is controlled genetically by factors that can vary between ecotypes.

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.

AT-rich sequences containing Arabidopsis-type telomere sequence and their chromosomal distribution in Pinus densiflora.

Japanese red pine Pinus densiflora has 2 n=24 chromosomes and after FISH-detection of Arabidopsis-type (A-type) telomere sequences, many telomere signals were observed on these chromosomes at interstitial and proximal regions in addition to the chromosome ends. These interstitial and proximal signal sites were observed as DAPI-positive bands, suggesting that the interstitial and proximal telomere signal sites are composed of AT-rich highly repetitive sequences. Four DNA clones (PAL810, PAL1114, PAL1539, PAL1742) localized at the interstitial telomere signals were selected from AluI-digested genomic DNA library using colony blot hybridization probed with A-type telomere sequences and characterized using FISH and Southern blot hybridization. The AT-contents of these selected four clones were 60.8-76.3%, and repeat units of the telomere sequence and degenerated telomere sequences were found in their nucleotide sequences. Except for two sites of PAL1114, FISH signals of the four clones co-localized with interstitial and proximal A-type telomere sequence signals. FISH signals a showed similar distribution pattern, but the patterns of signal intensity were different among the four clones. PAL810, PAL1539 and PAL 1742 showed similar FISH signal patterns, and the differences were only with respect to the signal intensity of some signal sites. PAL1114 had unique signals that appeared on chromosomes 7 and 10. Based on results of the Southern blot hybridization these four sequences are not arranged tandemly. Our results suggest that the interstitial A-type telomere sequence signal sites were composed of a mixture of several AT-rich repetitive sequences and that these repetitive sequences contained A-type telomere sequences or degenerated A-type telomere sequence repeats.

Characterization of a CENP-C homolog in Arabidopsis thaliana.

Centromere protein C (CENP-C) is a component of the kinetochore essential for correct segregation of sister chromatids in mammals. In Arabidopsis thaliana, a single-copy gene encoding a protein homologous to CENP-C has been found by homology in the whole-genome sequence. To investigate the CENP-C homolog (AtCENP-C), we cloned cDNAs by RT-PCR and determined its full-length coding sequence. Antibodies against the synthetic peptide for the C-terminal residues of AtCENP-C detected a polypeptide in Arabidopsis cell extracts on western blots. Immunofluorescence labeling with the antibodies and fluorescence in situ hybridization demonstrated clearly that AtCENP-C is present at the centromeric regions throughout the cell cycle.