This paper describes a fluorescence in situ hybridization (FISH) analysis of three different repetitive sequence families, which were mapped to mitotic metaphase chromosomes and extended DNA fibers (EDFs) of the two subspecies of rice (OrYza sativa), indica and japonica (2n = 2x = 24). The repeat families studied were (1) the tandem repeat sequence A (TrsA), a functionally non-significant repeat; (2) the [TTTA-GGG]n telomere sequence, a non-transcribed, tandemly repeated but functionally significant repeat; and (3) the 5S ribosomal RNA (5S rDNA). FISH of the TrsA repeat to metaphase chromosomes of indica and japonica cultivars revealed clear signals at the distal ends of twelve and four chromosomes, respectively. As shown in a previous report, the 17S ribosomal RNA genes (17S rDNA) are located at the nucleolus organizers (NORs) on chromosomes 9 and 10 of the indica cultivar. However, the japonica rice lacked the rDNA signals on chromosome 10. The size of the 5S rDNA repeat block, which was mapped on the chromosome 11 of both cultivars, was 1.22 times larger in the indica than in the japonica genome. The telomeric repeat arrays at the distal ends of all chromosome arms were on average three times longer in the indica genome than in the japonica genome. Flow cytometric measurements revealed that the nuclear DNA content of indica rice is 9.7% higher than that of japonica rice. Our data suggest that different repetitive sequence families contribute significantly to the variation in genome size between indica and japonica rice, though to different extents. The increase or decrease in the copy number of several repetitive sequences examined here may indicate the existence of a directed change in genome size in rice. Possible reasons for this phenomenon of concurrent evolution of various repeat families are discussed.
High-resolution fluorescence in situ hybridization (FISH) on interphase and pachytene nuclei, and extended DNA fibers enabled microscopic distinction of DNA sequences less than a few thousands of base pairs apart. We applied this technique to reveal the molecular organization of telomere ends in japonica rice (Oryza sativa ssp. japonica), which consist of the Arabidopsis type TTTAGGG heptameric repeats and the rice specific subtelomeric tandem repeat sequence A (TrsA). Southern hybridizations of DNA digested with Bal31 and EcoRI, and FISH on chromosomes and extended DNA fibers demonstrated that (1) all chromosome ends possess the telomere tandem repeat measuring 3-4 kb; (2) the subtelomeric TrsA occurs only at the ends of the long arms of chromosomes 6 and 12, and measure 6 and 10 kb, which corresponds to 231 and 682 copies for these sites, respectively; (3) the telomere and TrsA repeats are separated by at most a few thousands of intervening nucleotide sequences. The molecular organization for a general telomere organization in plant chromosomes is discussed.
SummaryChromosomes are super-molecules consisting of DNA, histone and chromatin proteins, which specifically appear within a cell at the cell division. We analyzed barley chromosomes by atomic force microscopy (AFM) to elucidate its structural basis. Mitotic chromosomes were taken from root tips of barley (Hordeum vulgare L., cv. Minorimugi) using the EMA (enzymatic maceration and air-drying) method after synchronization of cell cycle. Both the air-dried or critical point dried specimens were observed in air by a dynamic force mode. This observation technique enables to obtain three-dimensional image data on the surface structure of barley chromosomes at high resolution without any metal coating. The details of the higher order chromosomal structure such as chromatin fibers were clarified with the biological significance. Acidic treatment (e.g., acetic acid treatment) for removing proteins was useful to obtain clear images of basic chromosomal structure. Thus, it is concluded that the AFM has a great potential for investigation of molecular structures of chromosomes.Key words Atomic force microscopy (AFM), Chromosomal structure, Barley, Chromatin.The genetic information of the eukaryotic cell is stored in chromosomes. These structures are generated by condensation of chromatin fibers, which consist mainly of DNA and histone proteins. Numbers of papers have reported that modes of chemical modifications in DNA and histone proteins are closely related to the regulation of gene expression (reviewed by Rice and Allis 2001). They have revealed that regulation of the chromatin superstructure is closely related to the basic biological function such as transcription, replication, repair and DNA packaging through the cell cycle (Demeret et al. 2001). Primary control occurs through interactions between specific regulatory DNA sequences and large variety of transcription factors. Transcriptions are also regulated by which post-translational modification of histone proteins produces the structural change of chromatin (Struhl 1998); the phosphorylation of histones, for example, plays a role in inducing chromatin condensation (Dmitry et al. 2001). Acetylation of histone H4 is widely discussed as a factor, which causes the structural change of chromatin and serves for transcriptional regulation (Turner 2000, Wako et al. 2002, 2003.Viewed from the ultra-structural aspect, the basic structure of chromatin is the nucleosomes, which consist of histone octamer (H2A, H2B, H3, H4) and double-stranded DNA; the DNA is wound twice around the histone octamer to produce the "beads-on-a-string" form (reviewed by
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