Condensation of rye chromatin in somatic interphase nuclei of Ph1 and ph1b wheat

The Ph1 locus in hexaploid wheat (Triticum aestivum L.) enforces diploid-like behavior in the first metaphase of meiosis. To test the hypothesis that this chromosome pairing control is exercised by affecting the degree of chromatin condensation, the dispersion of rye chromatin in interphase nuclei in somatic tissues of wheat-rye chromosome translocations 1RS.1BL, 2RS.2BL, 2BS.2RL, 3RS.3DL and 5RS.5BL was compared in Ph1 and ph1b isogenic backgrounds. No significant differences in rye chromatin condensation that could be attributed to the Ph1 locus were detected. Regardless of the Ph1 status, each rye chromosome arm tested conformed to the general Rabl's orientation and occupied portions of the nuclei proportional to their length. Earlier observations that indicated the involvement of Ph1 locus in rye chromatin condensation in wheat could have been due either to specific loci on the studied 5RL rye arm that control the chromosome condensation process or to damage to the genetic system controlling chromatin condensation in the existing ph1b stocks of wheat. That damage might have been caused by homoeologous recombination and uneven disjunction of chromosomes from multivalents.     

Interphase chromosome arrangement in Anopheles atroparvus

The arrangement of chromosomes in interphase nuclei of Anopheles atroparvus has been inferred from an analysis of: 1. The early stages of mitosis as seen following Quinacrine staining, and 2. The reversible effects on the chromatin pattern obtained following the treatment of living cells with various NaCl solutions, and the following conclusions have been reached: (a) The chromatin is connected to the nuclear membrane, (b) Homologous chromosomes show close side-by-side somatic pairing, (c) The long arms of the sex chromosomes form a fluorescent peripheral body, (d) The autosomes are strongly reflexed at the centromeres, (e) The autosomal centromeric regions are polarized towards the peripheral body, (f) The telomeric regions of all the autosomes are closely apposed.--A ring-shaped pattern of interphase chromatin is constantly and reversibly induced by NaCl 0.15 to 0.18 M solutions.--These relationships indicate a peripheral arrangement of the interphase somatic complement.--The distribution of the chromosomes in polytene nuclei and at the beginning of meiosis resembles that suggested above for somatic interphase cells. This distribution may apply more widely in the Diptera.     

Effect of the pairing gene Ph1 on centromere misdivision in common wheat.

The cytologically diploid-like meiotic behavior of hexaploid wheat (i.e., exclusive bivalent pairing of homologues) is largely controlled by the pairing homoeologous gene Ph1. This gene suppresses pairing between homoeologous (partially homologous) chromosomes of the three closely related genomes that compose the hexaploid wheat complement. It has been previously proposed that Ph1 regulates meiotic pairing by determining the pattern of premeiotic arrangement of homologous and homoeologous chromosomes. We therefore assume that Ph1 action may be targeted at the interaction of centromeres with spindle microtubules--an interaction that is critical for movement of chromosomes to their specific interphase positions. Using monosomic lines of common wheat, we studied the effect of this gene on types and rates of centromere division of univalents at meiosis. In the presence of the normal two doses of Ph1, the frequency of transverse breakage (misdivision) of the centromere of univalent chromosomes was high in both first and second meiotic divisions; whereas with zero dose of the gene, this frequency was drastically reduced. The results suggest that Ph1 is a trans-acting gene affecting centromere-microtubules interaction. The findings are discussed in the context of the effect of Ph1 on interphase chromosome arrangement.     

Interphase chromosome order: A proposal

A model for the arrangement of chromosomes in interphase nuclei is proposed. The model assumes that interphase chromosomes have a Rabl orientation (a relic telophase arrangement). During interphase and prophase telomeres are attached to the nuclear envelope often in pairs. The association of telomeres, homologous or nonhomologous, is based on similarity of arm lengths and occurs at the time the nuclear envelope reforms. At this stage arm lengths will vary to some extent due to the amount of uncoiling etc. The sequence of chromosomes resulting from telomere-telomere pairing may vary among cells, but the number of arrangements will be restricted by arm length similarities.The ramifications of this model on melotic pairing, the constant attachment of chromosomes to some structure throughout the cell cycle, the distribution of genes within nuclei, and chromosome evolution are raised.     

Chromosome arrangement and nuclear architecture but not centromeric sequences are conserved between Arabidopsis thaliana and Arabidopsis lyrata

In contrast to the situation described for mammals and Drosophila, chromosome territory (CT) arrangement and somatic homologous pairing in interphase nuclei of Arabidopsis thaliana (n = 5) are predominantly random except for a more frequent association of the chromosomes bearing a homologous nucleolus organizer region. To find out whether this chromosome arrangement is also characteristic for other species of the genus Arabidopsis, we investigated Arabidopsis lyrata ssp. lyrata (n = 8), one of the closest relatives of A. thaliana. First, we determined the size of each chromosome and chromosome arm, the sequence type of centromeric repeats and their distribution between individual centromeres and the position of the 5S/45S rDNA arrays in A. lyrata. Then we demonstrated that CT arrangement, homologous pairing and sister chromatid alignment of distinct euchromatic and/or heterochromatic regions within A. lyrata interphase nuclei are similar to that in A. thaliana nuclei. Thus, the arrangement of interphase chromosomes appears to be conserved between both taxa that diverged about 5 million years ago. Since the chromosomes of A. lyrata resemble those of the presumed ancestral karyotype, a similar arrangement of interphase chromosomes is also to be expected for other closely related diploid species of the Brassicaceae family.     

Effect of the pairing gene Ph1 and premeiotic colchicine treatment on intra- and interchromosome pairing of isochromosomes in common wheat.

The analysis of the pattern of isochromosome pairing allows one to distinguish factors affecting presynaptic alignment of homologous chromosomes from those affecting synapsis and crossing-over. Because the two homologous arms in an isochromosome are invariably associated by a common centromere, the suppression of pairing between these arms (intrachromosome pairing) would indicate that synaptic or postsynaptic events were impaired. In contrast, the suppression of pairing between an isochromosome and its homologous chromosome (interchromosome pairing), without affecting intrachromosome pairing, would suggest that homologous presynaptic alignment was impaired. We used such an isochromosome system to determine which of the processes associated with chromosome pairing was affected by the Ph1 gene of common wheat-the main gene that restricts pairing to homologues. Ph1 reduced the frequency of interchromosome pairing without affecting intrachromosome pairing. In contrast, intrachromosome pairing was strongly reduced in the absence of the synaptic gene Syn-B1. Premeiotic colchicine treatment, which drastically decreased pairing of conventional chromosomes, reduced interchromosome but not intrachromosome pairing. The results support the hypothesis that premeiotic alignment is a necessary stage for the regularity of meiotic pairing and that Ph1 relaxes this alignment. We suggest that Ph1 acts on premeiotic alignment of homologues and homeologues as a means of ensuring diploid-like meiotic behavior in polyploid wheat.     

The effect of the wheat Ph1 locus on chromatin organisation and meiotic chromosome pairing analysed by genome painting

The Ph1 locus in wheat influences homo(eo)logous chromosome pairing. We have analysed its effect on the behaviour and morphology of two 5RL rye telosomes in a wheat background, by genomic in situ hybridisation (GISH), using rye genomic DNA as a probe. Our main objective was to study the effect of different alleles of the Ph1 locus on the morphology and behaviour of the rye telosomes in interphase nuclei of tapetal cells and in pollen mother cells at early stages of meiosis. The telosomes, easily detectable at all stages, showed a brightly fluorescing chromomere in the distal region and a constriction in the proximal part. These diagnostic markers enabled us to define the centromere and telomere regions of the rye telosomes. In the presence of functional copies of Ph1, the rye telosomes associated at pre-leptotene, disjoined and reorganised their shape at leptotene, and became fully homologously paired at zygotene – pachytene. In plants without functional alleles (ph1bph1b), the rye telosomes displayed an aberrant morphology, their premeiotic associations were clearly disturbed and their pairing during zygotene and pachytene was reduced and irregular. The Ph1 locus also influenced the behaviour of rye telosomes in the interphase nuclei of tapetal cells: in Ph1Ph1 plants, the rye telosomes occupied distinct, parallel-oriented domains, whereas in tapetal nuclei of ph1bph1b plants they were intermingled with wheat chromosomes and showed a heavily distorted morphology. The results shed new light on the effect of Ph1, and suggest that this locus is involved in chromosome condensation and/or scaffold organisation. Our explanation might account for various apparently contradictory and pleiotropic effects of this locus on both premeiotic associations of homologues, the regulation of meiotic homo(eo)logous chromosome pairing and synapsis, the resolution of bivalent interlockings and centromere behaviour. Received: 27 April 1998; in revised form: 5 August 1998 / Accepted: 11 August 1998     

Chromosome and DNA methylation dynamics during meiosis in the autotetraploid Arabidopsis arenosa

Variation in chromosome number due to polyploidy can seriously compromise meiotic stability. In autopolyploids, the presence of more than two homologous chromosomes may result in complex pairing patterns and subsequent anomalous chromosome segregation. In this context, chromocenter, centromeric, telomeric and ribosomal DNA locus topology and DNA methylation patterns were investigated in the natural autotetraploid, Arabidopsis arenosa. The data show that homologous chromosome recognition and association initiates at telomeric domains in premeiotic interphase, followed by quadrivalent pairing of ribosomal 45S RNA gene loci (known as NORs) at leptotene. On the other hand, centromeric regions at early leptotene show pairwise associations rather than associations in fours. These pairwise associations are maintained throughout prophase I, and therefore likely to be related to the diploid-like behavior of A. arenosa chromosomes at metaphase I, where only bivalents are observed. In anthers, both cells at somatic interphase as well as at premeiotic interphase show 5-methylcytosine (5-mC) dispersed throughout the nucleus, contrasting with a preferential co-localization with chromocenters observed in vegetative nuclei. These results show for the first time that nuclear distribution patterns of 5-mC are simultaneously reshuffled in meiocytes and anther somatic cells. During prophase I, 5-mC is detected in extended chromatin fibers and chromocenters but interestingly is excluded from the NORs what correlates with the pairing pattern.     

Centromere association is an unlikely mechanism by which the wheat Ph1 locus regulates metaphase I chromosome pairing between homoeologous chromosomes

Chiasmate pairing between homoeologous chromosomes at metaphase I (MI) of meiosis in wheat is prevented by the activity of the Ph1 locus on chromosome 5B. Several hypotheses have been proposed sharing the assumption that Ph1 regulates MI chromosome pairing by regulating centromere-mediated chromosome alignment before the onset of meiosis. To test the relevance of the putative predetermination of chromosome pairing at MI by the centromere-mediated chromosome association prior to meiosis, a 2BL.2RL homoeoisochromosome was constructed and its MI pairing was assessed in the presence and absence of the Ph1 locus. Although the 2BL and 2RL arms of the homoeoisochromosome paired with each other at MI in the absence of Ph1, they never paired with each other at MI in the presence of Ph1. Since the two arms were permanently associated in the homoeoisochromosome via a common centromere, it is unlikely that Ph1 predetermines MI pairing between homoeologous chromosomes solely by controlling premeiotic association of centromeres. These findings are consistent with the idea that Ph1 determines the chromosome pairing pattern at MI by scrutinizing homology across the entire chromosome.     

Chromosome territory arrangement and homologous pairing in nuclei of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> are predominantly random except for NOR-bearing chromosomes

Differential painting of all five chromosome pairs of Arabidopsis thaliana revealed for the first time the interphase chromosome arrangement in a euploid plant. Side-by-side arrangement of heterologous chromosome territories and homologous association of chromosomes 1, 3 and 5 (on average in 35–50% of nuclei) are in accordance with the random frequency predicted by computer simulations. Only the nucleolus organizing region (NOR)-bearing chromosome 2 and 4 homologs associate more often than randomly, since NORs mostly attach to a single nucleolus. Somatic pairing of homologous 100 kb segments occurs less frequently than homolog association, not significantly more often than expected at random and not simultaneously along the homologs. Thus, chromosome arrangement in Arabidopsis differs from that in Drosophila (characterized by somatic pairing of homologs), in spite of similar genome size, sequence organization and chromosome number. Nevertheless, in up to 31.5% of investigated Arabidopsis nuclei allelic sequences may share positions close enough for homologous recombination.Electronic Supplementary Material Supplementary material is available for this article at .     

Somatic association at interphase studied by giemsa banding technique

Somatic association of rye chromosomes has been studied by Giemsa banding technique at interphase in wheat-rye addition lines. Telomeres of the rye chromosomes appearing as chromocenters, showed close somatic association in disomic addition lines, but they were distributed at random in double monosomic additions. This demonstrates directly that somatic association of homologues at interphase is even closer in non-dividing nuclei than in metaphase cells, which had been investigated so far. The finding has relevance to the assumption that somatic association phenomena are a prerequisite to the meiotic pairing process.     

Stabilization and Localization of Xist RNA are Controlled by Separate Mechanisms and are Not Sufficient for X Inactivation

We examined the effect of cell cycle progression on various levels of chromosome organization in Drosophila. Using bromodeoxyuridine incorporation and DNA quantitation in combination with fluorescence in situ hybridization, we detected gross chromosomal movements in diploid interphase nuclei of larvae. At the onset of S-phase, an increased separation was seen between proximal and distal positions of a long chromsome arm. Progression through S-phase disrupted heterochromatic associations that have been correlated with gene silencing. Additionally, we have found that large-scale G1 nuclear architecture is continually dynamic. Nuclei display a Rabl configuration for only ∼2 h after mitosis, and with further progression of G1-phase can establish heterochromatic interactions between distal and proximal parts of the chromosome arm. We also find evidence that somatic pairing of homologous chromosomes is disrupted during S-phase more rapidly for a euchromatic than for a heterochromatic region. Such interphase chromosome movements suggest a possible mechanism that links gene regulation via nuclear positioning to the cell cycle: delayed maturation of heterochromatin during G1-phase delays establishment of a silent chromatin state.     

Random homologous pairing and incomplete sister chromatid alignment are common in angiosperm interphase nuclei

The chromosome arrangement in interphase nuclei is of growing interest, e.g., the spatial vicinity of homologous sequences is decisive for efficient repair of DNA damage by homologous recombination, and close alignment of sister chromatids is considered as a prerequisite for their bipolar orientation and subsequent segregation during nuclear division. To study the degree of homologous pairing and of sister chromatid alignment in plants, we applied fluorescent in situ hybridisation with specific bacterial artificial chromosome inserts to interphase nuclei. Previously we found in Arabidopsis thaliana and in A. lyrata positional homologous pairing at random, and, except for centromere regions, sister chromatids were frequently not aligned. To test whether these features are typical for higher plants or depend on genome size, chromosome organisation and/or phylogenetic affiliation, we investigated distinct individual loci in other species. The positional pairing of these loci was mainly random. The highest frequency of sister alignment (in >93% of homologues) was found for centromeres, some rDNA and a few other high copy loci. Apparently, somatic homologous pairing is not a typical feature of angiosperms, and sister chromatid aligment is not obligatory along chromosome arms. Thus, the high frequency of chromatid exchanges at homologous positions after mutagen treatment needs another explanation than regular somatic pairing of homologues (possibly an active search of damaged sites for homology). For sister chromatid exchanges a continuous sister chromatid alignment is not required. For correct segregation, permanent alignment of sister centromeres is sufficient.     

Fusion between interphase and mitotic plant protoplasts : Induction of premature chromosome condensation

Morphological changes in interphase nuclei were cytologically studied in heterophasic dinucleate cells formed by the fusion of mitotic and interphase plant protoplasts. Mitotic protoplasts were isolated from a partially synchronized suspension culture of wheat (Triticum monococcum). The mitotic cells were accumulated by colchicine after release of hydroxyurea block. Treatment of protoplast populations with polyethylene glycol-dimethyl sulphoxide solution resulted in metaphase-interphase fusion. Three hours after fusion, the appearance of chromosomes with single chromatid as well as of fragmented, pulverized chromatin in heterophasic cells indicated the induction of premature chromosome condensation (PCC) in somatic wheat cells. Condensation in interphase nuclei of mitotically inactive rice protoplasts was also detected after fusion with mitotic wheat protoplasts.     

Interphase chromosome structures of human cells

Major intermediates of chromosome condensation in erythroleukemia K562 cells are presented. Interphase chromatin structures became visible after reversal of permeabilization. Large-scale chromatin structures and the development of individual interphase chromosomes were observed by fluorescence microscopy. In the linear arrangement the following major intermediates of K562 chromatin condensation could be distinguished: (1) the most decondensed chromatin veil, (2) chromatin ribbon, (3) chromatin funnel, a new intermediate regarded as the earliest visible form of interphase chromosomes, (4) chromatin body, (5) 300 nm chromatin fiber, (6) u, v, or s forms of chromosomes, and (7) linear chromosomes. The observations made in nuclei of K562 cells conform to the model of helical coil chromosome condensation.     

The internal order of the interphase nucleus

Summary This paper has two parts. The first one is theoretical, whereas in the second, some experimenteal results are reported. Part 1: Theoretical Considerations. Comings' considerations on an ordered arrangement of chromatin in the interphase nucleus are used as a basis for further investigations and calculations in order to establish a preliminary model of the interphase nucleus. Information on the amount of DNA of a diploid human nucleus, on the degree of spiralization of chromatin threads found in electron microscopy, and measurements of salivary gland chromosomes was used to estimate the lengths of the entire interphase chromosomes. The number of fixing points-pores—was indirectly calculated proposing a model of an internal order of the chromatin threads. This number was found in concord with a direct calculation of the number of pores in the nuclear membrane based on results from electron microscopy. Part 2: Experimental Results and Discussion. In the second part of this study, an approach was made as to how to arrange chromosomes and chromosome segments in their proximity to each other. Results of cytogenetic studies of newborn babies and abortions, of cells from patients with Bloom's syndrome and Fanconi's anemia and normal cells treated with Mitomycin C and Trenimon, are thought to be informative under certain suppositions for the problem, which chromosome or chromosome parts are situated in proximity to each other. The symmetrical and equal interchanges seen, for example, in Bloom's syndrome are an indication of somatic pairing during the time of reunion. Therefore, the unequal interchanges in the same syndrome in which different chromosomes are involved should give evidence for proximity of nonhomologous chromosomes. Arguments for and against a temporal and spacial hypothesis for somatic pairing are discussed. The differing frequencies of chromosomes involved in Robertsonian translocations in man are informative for proximities of satellite regions at the nucleolus. Nucleolus and sex chromatin could be used as fixed points in a model of the interphase nucleus in which finally the absolute localization of the chromosomes will be discovered. The discussion points out promising methods for further investigations on the subject and mentions problems which could be attacked if the approach described here leads to a model of internal order in the interphase nucleus.This work was supported by the Deutsche Forschungsgemeinschaft within the Sonderforschungsbereich 35, Klinische Genetik.     

A cytochemical study of the nonhistone protein content of condensed and extended chromatin

Cytochemical techniques have been used to study the distribution of nonhistone proteins in sections of interphase nuclei and mitotic chromosomes. Condensed chromatin, including the heterochromatin of interphase nuclei from frog liver, and mitotic metaphase and anaphase chromosomes from bovine kidney, show little or no staining for nonhistone protein. Regions of frog liver nuclei which contain extended chromatin (euchromatin) stain intensely for nonhistone protein. These differences in nonhistone staining of condensed and extended chromatin support the suggestion that regions of condensed chromatin contain considerably less nonhistone protein than regions of extended chromatin. The results suggest further that there may be considerably less nonhistone protein associated with chromosomes and interphase heterochromatin than has been reported in most previous analyses of isolated chromatin and chromosome preparations.