We have characterized five genes encoding condensin components in Saccharomyces cerevisiae. All genes are essential for cell viability and encode proteins that form a complex in vivo. We characterized new mutant alleles of the genes encoding the core subunits of this complex, smc2-8 and smc4-1. Both SMC2 and SMC4 are essential for chromosome transmission in anaphase. Mutations in these genes cause defects in establishing condensation of unique (chromosome VIII arm) and repetitive (rDNA) regions of the genome but do not impair sister chromatid cohesion. In vivo localization of Smc4p fused to green fluorescent protein showed that, unexpectedly, in S. cerevisiae the condensin complex concentrates in the rDNA region at the G2/M phase of the cell cycle. rDNA segregation in mitosis is delayed and/or stalled in smc2 and smc4 mutants, compared with separation of pericentromeric and distal arm regions. Mitotic transmission of chromosome III carrying the rDNA translocation is impaired in smc2 and smc4 mutants. Thus, the condensin complex in S. cerevisiae has a specialized function in mitotic segregation of the rDNA locus. Chromatin immunoprecipitation (ChIP) analysis revealed that condensin is physically associated with rDNA in vivo. Thus, the rDNA array is the first identified set of DNA sequences specifically bound by condensin in vivo. The biological role of higher-order chromosome structure in S. cerevisiae is discussed.
We have determined the relationship between overall nuclear architecture, chromosome territories, and transcription sites within the nucleus, using three-dimensional confocal microscopy of well preserved tissue sections of wheat roots. Chromosome territories were visualized by GISH using rye genomic probe in wheat/rye translocation and addition lines. The chromosomes appeared as elongated regions and showed a clear centromere–telomere polarization, with the two visualized chromosomes lying approximately parallel to one another across the nucleus. Labeling with probes to telomeres and centromeres confirmed a striking Rabl configuration in all cells, with a clear clustering of the centromeres, and cell files often maintained a common polarity through several division cycles. Transcription sites were detected by BrUTP incorporation in unfixed tissue sections and revealed a pattern of numerous foci uniformly distributed throughout the nucleoplasm, as well as more intensely labeled foci in the nucleoli. It has been suggested that the gene-rich regions in wheat chromosomes are clustered towards the telomeres. However, we found no indication of a difference in concentration of transcription sites between telomere and centromere poles of the nucleus. Neither could we detect any evidence that the transcription sites were preferentially localized with respect to the chromosome territorial boundaries.
We report the identification of a family of sequences located by in situ hybridisation to the centromeres of all the Triticeae chromosomes studied, including the supernumerary and midget chromosomes, the centromeres of all maize chromosomes and the heterochromatic regions of rice chromosomes. This family of sequences (CCS1), together with the cereal genome alignments, will allow the evolution of the cereal centromeres and their sites to be studied. The family of sequences also shows homology to the CENP-B box. The centromeres of the cereal species and the proteins that interact with them can now be characterised.
We report the identification of a family of sequences located by in situ hybridisation to the centromeres of all the Triticeae chromosomes studied, including the supernumerary and midget chromosomes, the centromeres of all maize chromosomes and the heterochromatic regions of rice chromosomes. This family of sequences (CCS1), together with the cereal genome alignments, will allow the evolution of the cereal centromeres and their sites to be studied. The family of sequences also shows homology to the CENP-B box. The centromeres of the cereal species and the proteins that interact with them can now be characterised.
SummaryHexaploid wheat possesses 42 chromosomes derived from its three ancestral genomes. The 21 pairs of chromosomes can be further divided into seven groups of six chromosomes (one chromosome pair being derived from each of the three ancestral genomes), based on the similarity of their gene order. Previous studies have revealed that, during anther development, the chromosomes associate in 21 pairs via their centromeres. The present study reveals that, as a prelude to meiosis, these 21 chromosome pairs in hexaploid (and tetraploid) wheat associate via the centromeres into seven groups as the telomeres begin to cluster. This results in the association of multiple chromosomes, which then need to be resolved as meiosis progresses. The formation of the seven chromosome clusters now explains the occasional occurrence of remnants of multiple associations, which have been reported at later stages of meiosis in hexaploid (and tetraploid) wheat. Importantly, the chromosomes have the opportunity to be resorted via these multiple interactions. As meiosis progresses, such interactions are resolved through the action of loci such as Ph1, leaving chromosomes as homologous pairs.
Reduction in chromosome number and genetic recombination during meiosis require the prior association of homologous chromosomes, and this has been assumed to be a central event in meiosis. Various studies have suggested, however, that while the reduction division of meiosis is a universally conserved process, the pre-meiotic association of homologues differs among organisms. In the fruit fly Drosophila melanogaster, some somatic tissues also show association of homologues [1,2]. In the budding yeast Saccharomyces cerevisiae, there is some evidence for homologue association during the interphase before meiotic division [3,4], and it has been argued that such associations lead directly to meiotic homologue pairing during prophase I [5]. The available evidence for mammals suggests that homologous chromosomes do not associate in germ cells prior to meiotic prophase [6]. To study the occurrence of homologue pairing in wheat, we have used vibratome tissue sections of wheat florets to determine the location of homologous chromosomes, centromeres and telomeres in different cell types of developing anthers. Fluorescence in situ hybridization followed by confocal microscopy demonstrated that homologous chromosomes associate pre-meiotically in meiocytes (germ-line cells). Surprisingly, association of homologues was observed simultaneously in all the surrounding somatic tapetum cells. Homologues failed to associate at equivalent stages in a homologue recognition mutant. These results demonstrate that the factors responsible for the recognition and association of homologues in wheat act before the onset of meiotic prophase. The observation of homologue association in somatic tapetum cells demonstrates that this process and meiotic division are separable.
The amplified fragment length polymorphism (AFLP) technique was used to isolate DNA sequences present in the euploid wheat Chinese Spring but not in the Chinese Spring ph1b mutant (which has a deletion of the Ph1 gene, a suppressor of homoeologous chromosome pairing). The polymorphic DNA fragments identified by AFLP were then cloned, sequenced, and used to design two primer pairs. These primers were used in a PCR-based assay to specifically amplify products from the Chinese Spring euploid but not from the ph1b mutant. This PCR assay can be carried out from extracted genomic DNA or directly from alkaline-treated wheat leaves, and the reaction products can be scored on a plus-minus basis, making the screening amenable to automation. The reliability of the assay was tested using a F1-derived doubled-haploid population of 55 lines which segregate for the ph1b deletion. This PCR-screening technique is less time and labour consuming, and more accurate and reliable, than cytologically based conventional methods.
Homologue pairing mediates both recombination and segregation of chromosomes at meiosis I. The recognition of nucleic-acid-sequence homology within the somatic nucleus has an impact on DNA repair and epigenetic control of gene expression. Here we investigate interchromosomal interactions using a non-invasive technique that allows tagging and visualization of DNA sequences in vegetative and meiotic live yeast cells. In non-meiotic cells, chromosomes are ordered in the nucleus, but preferential pairing between homologues is not observed. Association of tagged chromosomal domains occurs irrespective of their genomic location, with some preference for similar chromosomal positions. Here we describe a new phenomenon that promotes associations between sequence-identical ectopic tags with a tandem-repeat structure. These associations, termed interchromosome trans-associations, may underlie epigenetic phenomena.
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