Tomographic distribution of acetylated histone H4 in plant chromosomes,nuclei and nucleoli

Root tip cells of broad bean (Vicia faba L. cv. ’Wase soramame’) and barley (Hordeum vulgare L. cv. ’Minorimugi’) were immunostained with antibodies specific for acetylated histone H4. With an antiserum that recognizes histone H4 acetylated at lysine-5, the nucleolar organizing region (NOR) in mitotic chromosomes was strongly labeled in both species. The broad bean had two signals in the metaphase and telophase chromosome complements and four signals in the prophase and anaphase chromosome complements, while the barley had four signals in the metaphase and telophase chromosome complements and eight signals in the prophase and anaphase complements. Five different patterns of signals were observed at interphase: in type I only nucleoli were wholly stained; in type II perinucleolar knob-like signals and/or fiber-like signals emanated from the nucleus; in type III aggregate signals appeared in the nucleolus; in type IV many small dot-like signals were distributed throughout the nucleus, except nucleoli; and in type V string-like or some granule-like signals appeared in the nucleoli. Type II was very similar to previous results by in situ hybridization with sense rDNA probes. Type III was similar to the patterns of DNA synthesis recognized as chromatin domains by anti-BrdU antibodies. Type V was very similar to the results of in situ hybridization with pTa71, rDNA probes and the appearance of the dense fibrillar components of the nucleolus. Received: 7 August 1996; in revised form: 16 September 1996 / Accepted: 16 September 1996     

Characterization of a splicing variant of plant Aurora kinase

Aurora kinases play a key role in chromosome segregation and cytokinesis. In plants, three Aurora kinases (AtAUR1-AtAUR3) have been identified in Arabidopsis thaliana. Here, we report an AtAUR2 splicing variant (AtAUR2S), which lacks the fourth exon encoding a part of the kinase domain of AtAUR2. AtAUR2S was shown to have lost its kinase activity to phosphorylate histone H3 at Ser10; however, it maintained its ability to bind to histone H3. The localization pattern of AtAUR2S was the same as that of AtAUR2. The findings suggest that AtAUR2S affects cell division by competing with AtAUR2.     

Molecular characterization of the soybean L-asparaginase gene induced by low temperature stress

L-asparaginase (EC 3.5.1.1) catalyzes the hydrolysis of the amide group of L-asparagine, releasing aspartate and NH4+. We isolated a low temperature-inducible cDNA sequence encoding L-asparaginase from soybean leaves. The full-length L-asparaginase cDNA, designated GmASP1, contains an open reading frame of 1,258 bp coding for a protein of 326 amino acids. Genomic DNA blotting and fluorescence in situ hybridization showed that the soybean genome has two copies of GmASP1. GmASP1 mRNA was induced by low temperature, ABA and NaCl, but not by heat shock or drought stress. E. coli cells expressing recombinant GmASP1 had 3-fold increased L-asparaginase activity. A possible function of L-asparaginase in the early response to low temperature stress is discussed.     

H1.X with different properties from other linker histones is required for mitotic progression

We report here the characterization of H1.X, a human histone H1 subtype. We demonstrate that H1.X accumulates in the nucleolus during interphase and is distributed at the chromosome periphery during mitosis. In addition, the results of fluorescence recovery after photobleaching indicate that the exchange of H1.X on and off chromatin is faster than that of the other H1 subtypes. Furthermore, RNA interference experiments reveal that H1.X is required for chromosome alignment and segregation. Our results suggest that H1.X has important functions in mitotic progression, which are different from those of the other H1 subtypes.     

Identification of a serum component that regulates cyclooxygenase-2 gene expression in cooperation with 4-hydroxy-2-nonenal

Atherosclerosis is a disorder of lipid metabolism as well as a chronic inflammatory disease. Cyclooxygenase-2 (COX-2), an inducible isoform responsible for high levels of prostaglandin production during inflammation and immune responses, mediates a variety of biological actions involved in vascular pathophysiology. We have previously shown that COX-2 gene expression is dramatically induced by a lipid-derived endogenous electrophile, 4-hydroxy-2-nonenal (HNE) (Kumagai, T., Matsukawa, N., Kaneko, Y., Kusumi, Y., Mitsumata, M., and Uchida, K. (2004) J. Biol. Chem. 279, 48389-48396). In the present study, based on the finding that HNE induced COX-2 expression only in the serum-containing media, we characterized a serum component essential for the HNE-induced COX-2 induction and found that low density lipoprotein (LDL) that had been denatured by freeze-thawing or oxidized LDL might be involved in the COX-2 induction. Moreover, we characterized the cellular events triggered by the combined stimulus of HNE and oxidized LDL and established that COX-2 induction is regulated by two sets of signaling mechanisms, one for the up-regulation of the scavenger receptor CD36 by HNE and one for the CD36-mediated COX induction by oxidized LDL. These findings represent a demonstration of a link between lipoprotein modification and activation of the inflammatory potential of macrophages.     

Genome and Chromosome Dimensions of Lotus japonicus

Lotus Japonicus , Miyakojima MG-20 and Gifu B-129. The genome sizes of Miyakojima and Gifu were determined as 472.1 and 442.8 Mbp, respectively. Both the accessions were diploid (2n=12) and six chromosomes were identified and characterized based on the condensation patterns and the locations of rDNA loci. The obvious polymorphism observed in the genome size and the chromosome morphology between the two accessions, revealed specific accumulation of heterochromatin in Miyakojima or elimination in Gifu. The chromosomes L. japonicus were numbered according to their length. A quantitative chromosome map was also developed by the imaging methods using the digital data of the condensation pattern. 45S rDNA loci were localized on chromosomes A and F, and 5S rDNA locus was localized on chromosome A by fluorescence in situ hybridization (FISH). Identification of the chromosome and genome sizes and development of the quantitative chromosome map represent significant contribution to the L. japonicus genome project as the basic information. Received 29 August 2000/ Accepted in revised form 17 October 2000     

Quantitative chromosome map of the polyploid Saccharum spontaneum by multicolor fluorescence in situ hybridization and imaging methods

Somatic chromosomes of a wild relative of sugarcane (Saccharum spontaneum L.) anther culture-derived clone (AP 85-361, 2n=32) were identified and characterized by computer-aided imaging technology and molecular cytological methods. The presence of four satellite chromosomes and four nearly identical chromosome sets suggests that the clone is a tetrahaploid with the basic number x=8. A quantitative chromosome map, or idiogram, was developed using image analysis of the condensation pattern (CP) at the prometaphase stage of somatic chromosomes. The 45S and 5S ribosomal RNA gene (rDNA) loci were simultaneously visualized by multi-color fluorescence in situ hybridization (McFISH) and precisely localized to the regions of 3p3.1 and 6q1.3 on the idiogram. The simultaneous visualization of two sets of four ribosomal RNA genes confirms tetraploidy of this clone. This conclusion is consistent with results of molecular marker mapping. The quantitative chromosome map produced will become the foundation for genome analyses based on chromosome identity and structure. Previously impossible identification of small chromosomes and untestable hypotheses about the polyploid nature of plants can now be settled with these two approaches of quantitative karyotyping and FISH.     

Development of a multistage classifier for a monitoring system of cell activity based on imaging of chromosomal dynamics.

BACKGROUND: Cell-based assays utilizing digital image cytometry yield multivariate sets of information measuring the efficacy of medicines/chemicals. The use of a HeLa cell line that expresses a GFP-Histone-H1 fusion protein further enhances the performance of these systems, avoiding the use of dyes that may have detrimental influence on cells. Aside from the mitotic index, the distribution of the cell-cycle phases during mitosis can be used as measures of drug/treatment efficacy. Quantification of these parameters, however, requires skill and is time consuming. The purpose of this research was therefore to create a classifier to be incorporated into a system that can automatically identify the cell-cycle phases in a given image. METHODS: Features based on the shape and texture of the chromosomal regions in images of live HeLa cells were measured and analyzed. Linear discriminant functions were calculated for the eight cell-cycle phases: interphase, prophase, prometaphase, metaphase, early anaphase, anaphase, telophase and cytokinesis. RESULTS: The multistage linear discriminant classifier developed had an average classification efficiency of 87.30%. CONCLUSION: We demonstrated the possibility of creating a classifier to discriminate between cell-cycle phases using shape and texture features of chromosomal regions. The classifier can be fused to an algorithm for image segmentation, forming a system to automatically and rapidly measure the aforementioned parameters. The results can then be collated to constitute an assay assessing the effects of a drug or treatment on mammalian cells.     

RAPD markers encoding retrotransposable elements are linked to the male sex in Cannabis sativa L.

Male-associated DNA sequences were analyzed in Cannabis sativa L. (hemp), a dioecious plant with heteromorphic sex chromosomes. DNA was isolated from male and female plants and subjected to random amplified polymorphic DNA analysis. Of 120 primers, 17 yielded 400 to 1500-bp fragments detectable in male, but not female, plants. These fragments were cloned and used as probes in gel-blot analysis of genomic DNA. When male and female DNA was hybridized with 2 of these male-specific fragments, MADC(male-associated DNA sequences in C. sativa)3 and MADC4, particularly intense bands specific to male plants were detected in addition to bands common to both sexes. The MADC3 and MADC4 sequences were shown to encode gag/pol polyproteins of copia-like retrotransposons. Fluorescence in situ hybridization with MADC3 and MADC4 as probes revealed a number of intense signals on the Y chromosome as well as dispersed signals on all chromosomes. The gel-blot analysis and fluorescence in situ hybridization results presented here support the hypothesis that accumulation of retrotransposable elements on the Y chromosome might be 1 cause of heteromorphism of sex chromosomes.