Functional dissection of in vivo interchromosome association in Saccharomyces cerevisiae.

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.

The condensin complex governs chromosome condensation and mitotic transmission of rDNA.

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.

Homologous chromosome pairing in wheat.

Bread wheat is a hexaploid (AABBDD, 2n=6x=42) containing three related ancestral genomes, each having 7 chromosomes, giving 42 chromosomes in diploid cells. During meiosis true homologues are correctly associated in wild-type wheat, but a degree of association of related chromosomes (homoeologues) occurs in a mutant (ph1b). We show that the centromeres are associated in non-homologous pairs in all floral tissues studied, both in wild-type wheat and the ph1b mutant. The non-homologous centromere associations then become homologous premeiotically in wild-type wheat in both meiocytes and the tapetal cells, but not in the mutant. In wild-type wheat, the homologues are colocalised along their length at this stage, but the telomeres remain distinct. A single telomere cluster (bouquet) is formed in the meiocytes only by the onset of leptotene. The sub-telomeric regions of the homologues associate as the telomere cluster forms. The homologous associations at the telomeres and centromeres are maintained through meiotic prophase, although, during leptotene, the two homologues and also the sister chromatids within each homologue are separate along the rest of their length. As meiosis progresses, first the sister chromatids and then the homologues associate intimately. In wild-type wheat, first the centromere grouping, then the bouquet disperse by the end of zygotene.

Transcription sites are not correlated with chromosome territories in wheat nuclei.

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.

A simple PCR-based method for scoring the ph1b deletion in wheat.

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.

Centromeric behaviour in wheat with high and low homoeologous chromosomal pairing.

Control of homoeologous chromosomal pairing in hexaploid wheat stems from a balance between a number of suppressor and promoter genes. This study used centromeric behaviour as a tool to investigate the mechanism. Fluorescent in situ hybridization employing centromeric and telomeric sequences as probes was applied to pollen mother cells of wheat and wheat/alien hybrids having different pairing gene combinations. It showed: association of centromeres during pre-meiotic interphase; decondensation of centromeric structure; sister chromatid disjunction of univalent chromosomes in homoeologous pairing situations at anaphase I; and centromeric stretching between univalent sister chromatids in wheat/rye hybrids deficient for pairing genes. The implications of these results are discussed.

Are rice chromosomes components of a holocentric chromosome ancestor?

Comparative genomics reveals that cereal genomes are composed of similar genomic building blocks (linkage blocks). By stacking these blocks in a unique order, it is possible to construct a single ancestral 'chromosome' which can be cleaved to give the basic structure of the 56 different chromosomes found in wheat, rice, maize, sorghum, millet and sugarcane. The borders of linkage blocks are defined by cereal centromeric and telomeric sites. However, a number of studies have shown that telomeric heterochromatin has neocentromeric activity, implying that linkage blocks are in fact defined by centromeric-like sites with conserved sequences. The structure of the ancestral cereal genome thus resembles a holocentric chromosome, which is the chromosome structure shared by the closest relatives of the Gramineae, the Cypericeae and Juncaceae.

Centromeric sites and cereal chromosome evolution.

Comparative genome analysis enables the sites of centromeres, telomeres and nucleolar organiser regions to be aligned with borders that define the sets of linked genes conserved across the cereal genomes. This provides a basis for studying cereal genome evolution.

Association of homologous chromosomes during floral development.

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.

A cereal centromeric sequence.

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.