Nuclear architecture in plants J.S. (Pat) Heslop-Harrison John Innes Centre for Plant Science, Norwich, UK Nuclei are dynamic structures that move through the mitotic cell cycle, are involved in differentiation, and divide and fuse during reproduction. The DNA contents of nuclei from different plants vary by 2500-fold. The design and structure of nuclei is, therefore, both flexible and versatile. Features relating to genome, chromosome, and maybe even gene localization during interphase are now emerging. At the chromosomal level, studies of scaffold associations and DNA sequence organization are indicating structures that impose nuclear architecture. Current Opinion in Genetics and Development 1992, 2:913-917
Introduction The first descriptions of the cell nucleus, more than a century ago, attempted to relate structure to function. Our understanding of the molecular biology of plants is now increasing rapidly, and the basis of interactions between DNA, enzymes, binding factors and expression modifiers is becoming known. However, a gap has appeared between this molecular knowledge, and cell and nuclear structure known from descriptive and morphological studies made using light or electron microscopy. Understanding nuclear architecture-- the subject of this r e v i e w - - b y combining molecular, cytological and quantitative approaches is critical to bridging the gap and thus enabling us to relate nuclear structure to evolution, recombination, gene expression and division.
Nuclear genome sizes The amount of DNA in the nuclei of different angiosperm plant species has a 2500-fold rang - - between 127 pg DNA (which c6rresponds to some 125 000Mb of DNA) per unreplicated haploid nucleus (lC DNA amount) in Fritillaria assyriaca, and 0.05 pg (50 Mb) in Cardamine amara [1,]. With increasing genome size, the proportion of highly repeated DNA increases from perhaps 20% to over 9596. All plants show similar patterns of growth, differentiation and development. However, the features of nuclear architecture vary over the range of DNA amounts, and may influence the expression of genes, DNA replication, cell division and recombination at meiosis. There are correlations between DNA amount and phenotypic characters ranging from cell or plant size and form, to minimum generation (life cycle) time, and the climatic zone in which the plant grows (see [1°]). The molecular genetics of Arabidopsis thaliana (Arabidopsis) is widely studied because of its small genome
size (100-150Mb), small plant size, fast generation time, and the extensive genetic knowledge, including many mutants, that has accumulated. Flow cytometry can demonstrate that the DNA contents of nuclei isolated from different Arabidopsis tissues vary widely because of systemic polyploidy or multiploidy [2-]. Most somatic cells are multiploid, with up to 16 times the 1 C DNA amount, although the inflorescences remain diploid (with unreplicated 2C, or replicated 4C DNA amounts). The extensive DNA variation, probably caused by chromosome endoreduplication [3"], seems to involve the complete genome, and is not selective. Galbmith et aL [2.] support the idea that differentiation often requires a minimal mass of nuclear DNA to maintain specific regulatory and functional states, as a consequence of which there is a minimum size of the nucleus related to DNA content, rather than directly to the number of structural genes. In dividing cells of species with high DNA contents, such as rye, there is often a gradient across the nucleus of the proportion filled by DNA, with the telomere end being more decondensed and having a much lower proportion of DNA than the centromere end [4]. Even in species with low DNA amounts, such as Arabidopsis, regions of the genome around the centromere tend to remain 'condensed', while the rest of the genome becomes extremely diffuse within the nucleus [3°]. Presumably the species with larger genomes have a lower proportion of transcriptionally active chromatin. Waterborg [5] has identified two histone variants, H3.1 and H3.2, and has compared their abundance in plants with a range of genome sizes. It has been suggested that H3.1 is a replicative histone variant associated with dividing cells, while H3.2 is a replacement variant, found in greater abundance in differentiated, transcriptionally active cells. Waterborg found that plants with smaller genomes had a higher proportion of the replacement variant, which presumably correlated with greater relative transcriptional activity of the DNA.
Abbreviation SAR--scaffold-associated region.
(~) Current Biology Ltd ISSN 0959-437X
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914 Genomesand evolution Packing of DNA into chromosomes
locus 1 cM is covered in 14 kb, as shown by transposon tagging strategies.
The structure of chromosomes has been examined to learn about chromatin fibre packing, supra-chromosomal organization of genes or repetitive DNA sequences and nuclear architecture. Using field emission scanning electron microscopy, Wanner et al. [6 o] imaged spread barley metaphase chromosomes. The centromeric constriction included a series of 30 run strands parallel to the long axis of the chromosome. Sister chromatids were clearly visible throughout their lengths, and tl~e coiling of the DNA from the 10-15 nm level of 'beadson-a-string' through the 30run solenoid, with further coiling of the solenoid before supercoiling into chromosomes, was described. The authors speculate about using the imaging system to investigate the spatial arrangement of chromosomes within the nucleus and its functional implications.
Understmlding the molecular architecture of large, highly methylated, plant genomes is becoming an essential complement to the description of small genomes. How are the gene locations related to vast amounts of non-coding DNA? In wheat and rye, unmethylated Noll sites mark gene-rich chromosomal regions, while peri-centromeric regions have few unmethylated sequences and have low gene densities [17-]. Unmethylated CpG sites can defiJle ends of blocks of families of repetitive DNA sequences [17--], and thus under-methylated CpG-rich regions can be used as genomic landmarks for regions rich in transcribed genes [18]. It seems likely that undennethylated sites, and particularly those sensitive to methylation changes, will mark genomic regions with active genes, and will be localized in domains within the interphase nucleus.
How are DNA sequences positioned and held within the nucleus? The nuclear scaffold is involved in maintaining chromosomal organization at interphase and was first described in animal nuclei, where scaffold-associated regions (SARs) of DNA clearly include origins of replication, centromeres and telomeres. Other SARs are linked to modifiers of gene expression. Plant scaffold proteins and associated DNA have now been described from pea [7"°], tobacco [8], soybean, potato and tomato. Both references discuss the possibility of scaffolds being related to the effects of the positions of genes with respect to other DNA sequences on their expression [9"]. Whether some gene co-suppression or trans-type interactions can be related to spatial locations of genes within the nucleus or with relationship to SARs or other DNA structures is not yet known.
Chromosomal organization In a year when the first 'plant' chromosome has been fully sequenced [10-], there has been much interest in the architecture and organization of chromosomes themselves. In other plants, this basic sequence information is years away, although the complete genome of Arabidopsis is now available in cloned form [11] and progress is rapid in joining contiguous sets of clones [12o.]. In larger genomes, DNA-DNA in situ hybridization is enabling the physical mapping of DNA sequences (restriction fragment length polymorphisms or genes) along chromosomes of rice [13] and barley [14o]. These data are highlighting the non-uniform distribution of genes along chromosomes. Neither genes nor homologous, meiotic, recombination events are uniformly distributed along plant chromosomes. This gives 'hot-spots' where recombination occurs with high frequency, and causes discrepancies in the distances between markers along the genetic and physical maps of the genomes [15]. Dooner [16] includes exceptional data: in maize, 1 cM of genetic length occupies on average some 2400 kb of DNA, whereas near the bronze
Genome behaviour in hybrid plants A wide range of interspecific and even intergeneric hybrids can be made in plants by sexual methods and cell fusion. Unlike most animal hybrids, the plant hybrids and the derived amphiploids, and chromosome addition or substitution ('alien') lines, are often viable. Much of the research on plmlt nuclear architecture has used these hybrids, since the behaviour of the individual alien chromosomes and parental genomes is straightforward to analyze. Hybrids with barley, Hordeum vulgare, show variable patterns of chromosome elimination depending on the parental genotypes. In some hybrid combinations, whole parental genomes are eliminated in the young F1 embryo, while others produce stable, diploid hybrid plants which flower. Linde-Laursen and von Bothmer [19 o] exan-tined the chromosomal constitution of 18 hypoploid, adult H. lechleri x barley plants. All hypoploid plants retained the nucleolus-forming barley chromosome 6, which is notable since previous studies (see [20]) have shown that the nucleolus-forming chromosomes tend to be lost first in other hybrids involving barley. Thus, there appears to be genotypic control over the elimination of specific chromosomes; individual chromosomes do not behave in a fashion random or independently of other chromosomes within the nucleus. Many hybrids between barley and other species show the phenomenon of parental genome separation at mitotic metaphase (see [2I]), and data increasingly suggest that parental chromosome complements constitute separate units [22]. How 'normal' is chromosome behaviour in sexual hybrids? The data indicate that the order of chromosome elimination is nonrandom and genome separation is actively maintained; it is unlikely that mechanisms would be active only in unusual hybrids, and the same genes perhaps affect aspects of chromosome behaviour and nuclear architecture in species, too. Bennett and Bennett [23"] have shown that Milium m o n t i a n u m is a wild, allotetraploid grass species of hybrid origin and that in mitotic metaphases,
Nuclear architecture in plants Heslop-Harrison 915 the two genomes are arranged concentrically throughout the development of the plant. The authors speculate that the probable genetic control of ancestral genome separation may have considerable underlying importance in plant evolution and speciation. In situ hybridization, using total genomic DNA as a probe, is now enabling chromosomes and genomes to be located not only at metaphase but throughout the cell cycle, Because probes and detection reagents might n o t penetrate uniformly throughout the interphase nucleus, initial investigations required reconstructions of probed consecutive sections. In barley x Secale a f r i c a n u m , it was evident that the two sets of seven chromosomes from each parent were spatially separated, not only at metaphase but throughout the cell cycle, at least in cells from near the root meristem [24..]. Some variability in patterns was found between cells, indicating that the chromosome location is maintained, or perhaps reinforcecl, by an active mechanism, but may be influenced by developmental or environmental factors.
At anaphase in barley x S. a f r i c a n u m hybrids, the two parental genomes separate simultaneously. In contrast, in another hybrid, barley x 14. bulbosum, which also shows genome separation throughout the cell cycle, the chromosomes from the H. b u l b o s u m genome lag during division [25]. Thus, organization of the nucleus during division, and spatial locations of whole sets of chromosomes, can be modulated during the cell cycle, but the genetic control of this behaviour is evidenced by the contrasting behaviour in different hybrids.
Meiotic reorganization Examination of locations of single pairs of homologous chromosomes in somatic interphase nuclei confirms the impression gained from metaphase analysis that homologues are not paired during most of the cell cycle [26-.]. In a hybrid plant, metaphase analysis indicates that homologous chromosomes, which may pair during the early ~tages of meiosis, are still apart at the last somatic metaphase before meiosis [27"]. Thus, a major physical reorganization of the nucleus occurs during the extended last pre-meiotic interphase and before pachytene of meiosis. In the human [28], yeast [29..], and some cereals (T. Schwarzacher, unpublished data), we can begin to follow whole chromosomes or chromosome arms during the pre-meiotic interphase and meiotic prophase by in situ hybridization of probes that paint whole chromosomes or chromosome arms. In the next few years, we can look forward to a detailed analysis of when chromosome pairing occurs, where it is initiated along the chromosomes, and when and how recombination takes place. Perhaps we shall also find the exact role of synaptonemal complex: evidence from yeast mutants indicates that the structure is independent of recombination [30]. In the cereals, the physical clustering of genes near the ends of chromosomes is not evident on genetic maps
because most recombination occurs in this region. Do chromosomes condense differentially during meiosis to reflect the recombination sites? At least by pachytene, markers of heterochromatin and rDNA seem to be in the same positions along chromosome arms as at mitosis [31"]. It is noteworthy that the sizes of rDNA sites, as measured by in situ hybridization, to occupy much more of the chromosome length at mitosis than at meiosis; the meiotic measurements correlate closely with the proportion of the genome that is rDNA.
The nucleolus The nucleolus is the most conspicuous feature of nuclear architecture in the light microscope. The remarkable controversy over its structure, and, in particular, where the transcribing genes lie, continues. Jordan [32] has presented seven models, each supported by some evidence concerning the internal structure, organization and activity of the body. High-resolution in situ hybridization of rDNA probes to electron microscope preparations has n o w enabled re-interpretation of light microscope data in both human [33] and plant nucleoli [34.]. The similarity between the morphologically distinct nucleoli is striking, with both showing active, diffuse genes near the dense fibrillar component, although there are substantial differences in the localization of condensed, unexpressed genes even in species as closely related as wheat and rye [34°]. Unmethylated cytosine residues at specific sites within the rDNA repeat unit correlate with gene expression and perturbation of chromatin structure [35"]. A cytological stud), of the nucleolus is indeed close to being associated with a molecular investigation of gene structure.
Conclusions Molecular cytogenetic studies using in situ hybridization at light and electron microscope levels are now allow. ing the structure of the interphase nucleus to be directly probed. For the first time we can localize a particular DNA sequence within any nucleus at any developmental stage. As a result, relationships between nuclear architecture and function, evolution, genetics and molecular biology are rapidly emerging. Comparative studies, covering a range in nuclear DNA contents and using hybrids, are enabling us to describe general features of nuclear and chromosomal architecture in plants.
Acknowledgements I am grateful to Trude Schwarzacher, Andrew keitch, Kesara Anamthawat-J6nsson, Ilia Leitch, Gill Harrison and Shi Min for their enormous contributions to my research program. I thank them and Mikael Roose for helpful comments, and many other authors for
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Genomes and evolution pre-prints. I thank BP Venture Research and AFRC for supporting my research programme.
to become methylated and inactivated, and gene suppression is correlated closely with methylation. Stable expression of inserted genes will require the absence of modifier sequences causing such suppression. 10,
OLIVERSG, VAN DER AART QJM, AGOSTON1-CARBONEML, AIGLE M, ALBERGHINA L, ALEXANDRAKI D, ANTOINE G, ANWAR R, BALLESTAJPG, BENIT P, ET AL:The Complete DNA Sequence of Yeast C h r o m o s o m e IIi. Nature 1992, 357:38-46. The first complete sequence of a eukaryotic chromosome. Many, ORFs of unknown function were identified. The comparison of the physical (sequence) map with the genetic map shows regions of increased and decreased meiotic recombination. .o
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest •, of outstanding interest BENNETTMD, SMITH JB: Nuclear DNA A m o u n t s in An. giosperms. Phil Trans R Soc Lond [Biol] 1991, 334:309--345. This is a compendium of genome sizes, adding to three previous collected lists by the same authors, and increasing the number of plant species where DNA amounts have been measured to over 1500 (of the 250 000 angiospeml species known). It is of wide use to cell and molecular biologists (as well as ecologists and taxonomists) who are interested in genome analysis and tile consequences of absolute nuclear genome size.
11.
1.
2. •
GAmP, ArrH DW, HARmNS KR, KNAPP S: Systemic Endopolyploidy in Arabidopsis thaliana. Plant Ph3~iol 1991, 96:985-989. Endoreduplication is found in all somatic tissues of tile species except for the intlorescences. The concept of a need for a minimal mass of nuclear DNA to maintain specific regulator3, and functional states is extensively discussed. 3. •
MALUSZYNSKA J, HESLOP-HARRISONJS: Localization of Tandemly Repeated DNA Sequences in Arabidopsis thaliana. Plant J 1991, 1:159-166. Fluorescent staining methods enable the vet3 , small chromosomes of Arabidopsis to be easily studied. In situ hybridization is used to map the rDNA sequences (8% of the genome) to two chromosome pairs. A centromeric repeat sequence, about 1.5% of the genome, is mapped to all five chromosome pairs. 4.
ANATOFIAWAT-JONSSONK, HESLOP-HARRLSONJS: Centromeres, Telomeres and Chromatin in t h e Interphase Nucleus of Cereals. Caryologia 1990, 43:205-213.
5,
WATERBORGJH: Existence of T w o Histone H3 Variants in Dicotyledonous Plants and Correlation Between their Acetylation and Plant G e n o m e Size. Plant Mol Biol 1992, 18:181-187.
6.
WANNERG, FORMANEK H, MARTIN R, HERRbtANNRG: High Res-
•
olution Scanning Electron Microscopy of Plant Chromosomes. Cbromosoma 1991, 100:103-109. This paper reports field emission scanning electron micrographs of spread barley chromosomes, imaging structures down to 10nm. A detailed technique is presented, and the method has great potential for anab/sis of chromosome condensation and decondensation. 7. •,
SLATI'ERRE, DUPREE P, GRAYJC: A Scaffold-associated DNA Region is Located D o w n s t r e a m o f the Pea Plastocyanin Gene. Plant Cell 1991, 3:1239-1250. Not only, detailed and careful work describing the first isolation and sequencing of a SAR DNA region from plants, but an excellent introduction that puts the research findings into the context of nuclear architecture and the related animal work. 8.
HALL G, ALLENGC, LOER DS, THOMPSON WF: Nuclear Scaffolds and Scaffold-attachment Regions in Higher Plants. Proc Natl Acad Sci USA 1991, 88:9320-9324.
MATZKE /VIA, MATZKE AJM: Differential Inactivation and Methylation of a T r a n s g e n e in Plants by T w o Suppressor Loci Containing Homologous Sequences. Plant Mol Biol 1991, 16:821-830. In doubly transformed tobacco plants, an inserted marker gene can be unexpectedly inactivated when a second and different gene is inserted in an unlinked site. Here, gene constructs are used that give complete or partial suppression of the first locus in transformed plants. The second transforming DNA can, depending on the construct, cause the first gene
Gva~ E, SOMERVIH.E C: Construction and Characterization of a Yeast Artificial C h r o m o s o m e Library of Arabtdopsis w h i c h is Suitable for C h r o m o s o m e Walking. Mol Gen Genet 1991, 226:484--490.
SCHMIDT R, DEAN C: Physical Mapping of t h e Arabidopsis thaliana Genome. In Genome Analysis: Strategies for Pto,sical Mapping. Edited by Davies KR. Cold Spring Harbor: Cold Spring Harbor Press; 1992, in press. A description of the strategies for, and progress in, generating physical maps from c h r o m o s o m e - ~ l k i n g experiments ,and making an overlapping clone library in this model plant species. 12. ..
13.
GUSTAFSONJP, DILLEJE: T h e Chromosomal Location of Oryza Sativa Recombination Linkage Groups 1,2. Proc Natl Acad Sci USA 1992, 89:8646-8650.
14.
LEITCH IJ, HESLOP-HARRISON JS: Physical Mapping of the 18S-5.8S--26S rRNA G e n e s in Barley by in Situ Hybridization. Genome 1992, in press. High-resolution in situ hybridization is able to map five sites of rRNA genes in barley, compared to the two mapped by other methods. There are four sites of 5S rRNA genes, rather than the one mapped previously. The cx-amylase gene is distal on c h r o m o s o m e 5H. Comparison of the physical locations of these genes with the genetic locations shows that most recombination occurs at tile ends of chromosomes. •
15.
HESLOP-HARRISONJS: T h e Molecular Cytogenetics of Plants. J Cell Sci 1991, 100:15-21.
16.
DOONERHK: Genetic Fine Structure of t h e Bronze Locus in Maize. Genetics 1986, 113:1021-1036.
MOOREG, CHEUNG W, FOOTE T, GALE M, KOEBNER R, LEITCH AR, LEITCH lJ, MONEY T, STANCOMBE P, YANO M, FLAVELLPc Key Features of Cereal G e n o m e Organisation as Revealed by the Use of Cytosine Methylation-sensitive Restriction Endonucleases. Genomics 1992, in press. An extensive paper that brings together molecular, genetical and cytological experiments to begin to show how large genomes are organized, where repetitive DNA is localized, and how different sequence families are spatially related to each other along the chromosomes. The data provide a basis for targeted gene isolation and manipulation of large genomes. 17. •e
18.
CHEUNGWY, MOORE G, MONEY TA, GALE MD: Hpall Library Indicates 'Methylation-free Islands' in W h e a t and Barley. Theor Appl Genet 1992, 84:739-746.
19. .
LINDE-LAURSENI, VON BOTHMER R: Barley C h r o m o s o m e 6 Rerained Specifically in Hypoploid Hybrids of a C h r o m o s o m e eUminating Interspecific Cross. Barley Genet 1991, 6:77-79. Loss of chromosomes occurs during cell division. This reports the preferential retention of o n e c h r o m o s o m e in hybrids, indicaUng that mechanisms ensure segregation of particular chromosomes. 20.
FINCHRA, BENNETF MD: T h e M e c h a n i s m of Somatic Chromos o m e Elimination in Hordeum. Kew Chromosome Conference. Edited by Brandham PE, Bennett MD. London: Allan & Unwin; 1983, 2:147-154.
21.
HESLOP-HARRISONJS, BENNETT MD: Nuclear Architecture in Plants. Trends Genet 1990, 6:401-405.
22.
LINDE-LAURSENI, JENSEN J: G e n o m e and C h r o m o s o m e Disposition at Somatic Metaphase in a H o r d e u m x Psathyrostachys Hybrid. Heredity 1991, 66:203-210.
9. •
Nuclear architecture in plants 23. •
BENNETI"ST, BENNETI" MD: Spatial Separation of Ancestral G e n o m e s in the Wild Grass M i l i u m m o n t i a n u m Pari. Ann Bot 1992, 70:111-118. M. montianum is a wild allotetraploid grass. In both mitotic and meiotic nuclei, the ancestral genomes tended to lie apart in spread metaphase preparations. It is suggested that the nuclear organization is under genetic control and may facilitate independent behaviour of the genomes in the polyploid, leading to speciation. 24. ••
LEITCH AR, SCHWARZACHERT, MOSGOLLER W, BENNETT MD, HESLOP-HARRISONJS: Parental G e n o m e s are Separated T h r o u g h o u t the Cell Cycle in a Plant Hybrid. Cbromosoma 1991, 101:206-213. In situ hybridization is used to localize whole genomes in a sexual hybrid plant (barley x S. africanam, both n = 7) on both spread and sectioned preparations. Reconstructions show that the one chromosome set is located around the nucleolus, while the other occupies one, two or three domains in the peripheral region of the nucleus. 25.
ANAMTHAWAT-JON~SON K, SCHWARZACHERT, HESLOP-HARRISONJS: Behavior of Parental G e n o m e s in H o r d e u m vulgate x H. bulbosum. J Hered 1992, in press.
26. ••
SCHWARZACHER T, ANAMTHAWAT-JONSSONK, HARRISON GE, ISt~t AK&IR,JIA JZ, KING IP, LEITCH AR, MILLER TE, READER SM, ROGERS WJ, bY/"AL: G e n o m i c in Situ Hybridization to Identify Alien C h r o m o s o m e s and C h r o m o s o m e Segments in Wheat. 77Jeor Appl Genet 1992, 84:778-786. Total genomic DNA can be used as a probe for in situ hybridization to identify chromosomes and chromosome segments introduced from many different ('alien') species into wheat. The method also shows the locations of the alien chromosomes at interphase; in diploid lines, the alien chromosomes are not associated but lie in different domains. 27.
SCtTWARZACHERT, HESLOP.HARRISON JS, ANAMTHAWAT-JONSSON K, FINCH RA, BENNE'VI" MD: Parental G e n o m e Separation in Reconstructions of Somatic and Premeiotic Metaphases of H o r d e u m vulgare x H. bulbosum. J Cell Sci 1992, 101:13-24. Serial section reconstructions enable analysis of the three-dinaensional location and identification of each chromosome. The parental genomes are apart in root-tip cells and pollen mother cells at the last mitotic division before meiosis. The authors speculate that the nucleus is reorganized physically to align homoeologous or, in species, homologous, chromosomes, after this mitosis, but before meiosis.
•
28.
29. •,
GOLD/~L,XNASH, HULTEN MA: C h r o m o s o m e in Situ Suppression Hybridization in H u m a n Male Meiosis. J Med Genet 1992, 29:98-102.
SCHERTHANH, LOIDL J, SCHUSTER T, SCHWEIZER D: Meiotic C h r o m o s o m e Condensation and Pairing in S. cerevisiae Studied by C h r o m o s o m e Painting. Chromosoma 1992, 101:590-595. Movements of homologous c h r o m o s o m e segments were followed during early meiotic prophase by in situ hybridization of pools of DNA
Heslop-Harrison
clones. The chromatin was reported to condense, and homologous segments align, before synaptonemal complex formation, which has major implications for models of chromosome recognition, recombination and pairing. 30.
HABERJE, LEUNGW-Y, BORTS R.H, LICHTEN M: The Frequency of Meiotic Recombination in Yeast is I n d e p e n d e n t of t h e N u m b e r and Position of Homologous Donor Sequences: Implications for C h r o m o s o m e Pairing. Proc Natl Acad Sci USA 1991, 88:1120-1124.
AtBINI SM, SCHWAP,ZACHER T: I n Sign Localization of T w o Repetitive DNA Sequences to Surface-spread Pachytene C h r o m o s o m e s of Rye. Genome 1992, 35:551-559. Meiotic prophase nuclei were surface-spread and used for in situ hybridization with the rDNA sequence and a heterochromatin sequence. The DNA associated with the bivalent axes (and synaptonemal complex) was stained strongly with DNA fluorochromes and probe hybridization occurred along the taxes. Tile method will be valuable for high-resolution physical mapping of DNA sequences and investigation of the locations of defined DNA sequences involved in pairing and recombination. 31. •
32.
JORDANEG: Interpreting Nucleolar Structure: W h e r e are the Transcribing Genes? J Cell Sci 1991, 98:437-442.
33.
WACHTLER F, MOSGOLLERW, SCHWARZ.AGHERHG: Electron Microscopic in Situ Hybridization and Autoradiography: Localization and Transcription of rDNA in H u m a n Lymphocyte Nucleoli. E.xp Cell Res 1990, 187:346-348.
34. •
I.ErrCHAR, MOSGOLLERW, SHI M, HESLOP-HARRISONJS: Differ. e n t Patterns of rDNA Organization at lnterphase in Nuclei of W h e a t and Rye. J Cell Sci 1992, 101:751-757. In situ hybridization, using light and electron microscopy is used to 1o. cate rRNA genes within interphase nucleoli. In wheat, condensed rDNA foci are visible within the nucleolus, with fine threads between them called fragmented decondensation. Only rye has the diffuse sites, which are likely to represent active rRNA genes. Thus, the organization of gene expression is fundamentally different in the two related species. 35. .•
SARDANAR, FLAVELLRB, O'DELL M: Correlation b e t w e e n t h e Size of the Intergenic Regulatory Region, t h e Status of Cytosine Methylation of rRaNA G e n e s and Nucleolar Expression in Wheat. Mol Gen Genet 1992, in press. Relative nucleolus organizer (rDNA site) activity, is correlated with the number of repeats of a 135 bp sequence upstream of the gene promoter. Active loci include more gene repeat units where there are unmethylated cytosines at specific sites in the promoter and in the 135 bp repeat, and in which chromatin structure is perhaps altered.
JS Heslop-Harrison, Department of Cell Biology, John lnnes Centre for Plant Science Research, Colney Lane, Norwich NR4 7UJ, UK.
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Introduction The first descriptions of the cell nucleus, more than a century ago, attempted to relate structure to function. Our understanding of the molecular biology of plants is now increasing rapidly, and the basis of interactions between DNA, enzymes, binding factors and expression modifiers is becoming known. However, a gap has appeared between this molecular knowledge, and cell and nuclear structure known from descriptive and morphological studies made using light or electron microscopy. Understanding nuclear architecture-- the subject of this r e v i e w - - b y combining molecular, cytological and quantitative approaches is critical to bridging the gap and thus enabling us to relate nuclear structure to evolution, recombination, gene expression and division.
Nuclear genome sizes The amount of DNA in the nuclei of different angiosperm plant species has a 2500-fold rang - - between 127 pg DNA (which c6rresponds to some 125 000Mb of DNA) per unreplicated haploid nucleus (lC DNA amount) in Fritillaria assyriaca, and 0.05 pg (50 Mb) in Cardamine amara [1,]. With increasing genome size, the proportion of highly repeated DNA increases from perhaps 20% to over 9596. All plants show similar patterns of growth, differentiation and development. However, the features of nuclear architecture vary over the range of DNA amounts, and may influence the expression of genes, DNA replication, cell division and recombination at meiosis. There are correlations between DNA amount and phenotypic characters ranging from cell or plant size and form, to minimum generation (life cycle) time, and the climatic zone in which the plant grows (see [1°]). The molecular genetics of Arabidopsis thaliana (Arabidopsis) is widely studied because of its small genome
size (100-150Mb), small plant size, fast generation time, and the extensive genetic knowledge, including many mutants, that has accumulated. Flow cytometry can demonstrate that the DNA contents of nuclei isolated from different Arabidopsis tissues vary widely because of systemic polyploidy or multiploidy [2-]. Most somatic cells are multiploid, with up to 16 times the 1 C DNA amount, although the inflorescences remain diploid (with unreplicated 2C, or replicated 4C DNA amounts). The extensive DNA variation, probably caused by chromosome endoreduplication [3"], seems to involve the complete genome, and is not selective. Galbmith et aL [2.] support the idea that differentiation often requires a minimal mass of nuclear DNA to maintain specific regulatory and functional states, as a consequence of which there is a minimum size of the nucleus related to DNA content, rather than directly to the number of structural genes. In dividing cells of species with high DNA contents, such as rye, there is often a gradient across the nucleus of the proportion filled by DNA, with the telomere end being more decondensed and having a much lower proportion of DNA than the centromere end [4]. Even in species with low DNA amounts, such as Arabidopsis, regions of the genome around the centromere tend to remain 'condensed', while the rest of the genome becomes extremely diffuse within the nucleus [3°]. Presumably the species with larger genomes have a lower proportion of transcriptionally active chromatin. Waterborg [5] has identified two histone variants, H3.1 and H3.2, and has compared their abundance in plants with a range of genome sizes. It has been suggested that H3.1 is a replicative histone variant associated with dividing cells, while H3.2 is a replacement variant, found in greater abundance in differentiated, transcriptionally active cells. Waterborg found that plants with smaller genomes had a higher proportion of the replacement variant, which presumably correlated with greater relative transcriptional activity of the DNA.
Abbreviation SAR--scaffold-associated region.
(~) Current Biology Ltd ISSN 0959-437X
913
914 Genomesand evolution Packing of DNA into chromosomes
locus 1 cM is covered in 14 kb, as shown by transposon tagging strategies.
The structure of chromosomes has been examined to learn about chromatin fibre packing, supra-chromosomal organization of genes or repetitive DNA sequences and nuclear architecture. Using field emission scanning electron microscopy, Wanner et al. [6 o] imaged spread barley metaphase chromosomes. The centromeric constriction included a series of 30 run strands parallel to the long axis of the chromosome. Sister chromatids were clearly visible throughout their lengths, and tl~e coiling of the DNA from the 10-15 nm level of 'beadson-a-string' through the 30run solenoid, with further coiling of the solenoid before supercoiling into chromosomes, was described. The authors speculate about using the imaging system to investigate the spatial arrangement of chromosomes within the nucleus and its functional implications.
Understmlding the molecular architecture of large, highly methylated, plant genomes is becoming an essential complement to the description of small genomes. How are the gene locations related to vast amounts of non-coding DNA? In wheat and rye, unmethylated Noll sites mark gene-rich chromosomal regions, while peri-centromeric regions have few unmethylated sequences and have low gene densities [17-]. Unmethylated CpG sites can defiJle ends of blocks of families of repetitive DNA sequences [17--], and thus under-methylated CpG-rich regions can be used as genomic landmarks for regions rich in transcribed genes [18]. It seems likely that undennethylated sites, and particularly those sensitive to methylation changes, will mark genomic regions with active genes, and will be localized in domains within the interphase nucleus.
How are DNA sequences positioned and held within the nucleus? The nuclear scaffold is involved in maintaining chromosomal organization at interphase and was first described in animal nuclei, where scaffold-associated regions (SARs) of DNA clearly include origins of replication, centromeres and telomeres. Other SARs are linked to modifiers of gene expression. Plant scaffold proteins and associated DNA have now been described from pea [7"°], tobacco [8], soybean, potato and tomato. Both references discuss the possibility of scaffolds being related to the effects of the positions of genes with respect to other DNA sequences on their expression [9"]. Whether some gene co-suppression or trans-type interactions can be related to spatial locations of genes within the nucleus or with relationship to SARs or other DNA structures is not yet known.
Chromosomal organization In a year when the first 'plant' chromosome has been fully sequenced [10-], there has been much interest in the architecture and organization of chromosomes themselves. In other plants, this basic sequence information is years away, although the complete genome of Arabidopsis is now available in cloned form [11] and progress is rapid in joining contiguous sets of clones [12o.]. In larger genomes, DNA-DNA in situ hybridization is enabling the physical mapping of DNA sequences (restriction fragment length polymorphisms or genes) along chromosomes of rice [13] and barley [14o]. These data are highlighting the non-uniform distribution of genes along chromosomes. Neither genes nor homologous, meiotic, recombination events are uniformly distributed along plant chromosomes. This gives 'hot-spots' where recombination occurs with high frequency, and causes discrepancies in the distances between markers along the genetic and physical maps of the genomes [15]. Dooner [16] includes exceptional data: in maize, 1 cM of genetic length occupies on average some 2400 kb of DNA, whereas near the bronze
Genome behaviour in hybrid plants A wide range of interspecific and even intergeneric hybrids can be made in plants by sexual methods and cell fusion. Unlike most animal hybrids, the plant hybrids and the derived amphiploids, and chromosome addition or substitution ('alien') lines, are often viable. Much of the research on plmlt nuclear architecture has used these hybrids, since the behaviour of the individual alien chromosomes and parental genomes is straightforward to analyze. Hybrids with barley, Hordeum vulgare, show variable patterns of chromosome elimination depending on the parental genotypes. In some hybrid combinations, whole parental genomes are eliminated in the young F1 embryo, while others produce stable, diploid hybrid plants which flower. Linde-Laursen and von Bothmer [19 o] exan-tined the chromosomal constitution of 18 hypoploid, adult H. lechleri x barley plants. All hypoploid plants retained the nucleolus-forming barley chromosome 6, which is notable since previous studies (see [20]) have shown that the nucleolus-forming chromosomes tend to be lost first in other hybrids involving barley. Thus, there appears to be genotypic control over the elimination of specific chromosomes; individual chromosomes do not behave in a fashion random or independently of other chromosomes within the nucleus. Many hybrids between barley and other species show the phenomenon of parental genome separation at mitotic metaphase (see [2I]), and data increasingly suggest that parental chromosome complements constitute separate units [22]. How 'normal' is chromosome behaviour in sexual hybrids? The data indicate that the order of chromosome elimination is nonrandom and genome separation is actively maintained; it is unlikely that mechanisms would be active only in unusual hybrids, and the same genes perhaps affect aspects of chromosome behaviour and nuclear architecture in species, too. Bennett and Bennett [23"] have shown that Milium m o n t i a n u m is a wild, allotetraploid grass species of hybrid origin and that in mitotic metaphases,
Nuclear architecture in plants Heslop-Harrison 915 the two genomes are arranged concentrically throughout the development of the plant. The authors speculate that the probable genetic control of ancestral genome separation may have considerable underlying importance in plant evolution and speciation. In situ hybridization, using total genomic DNA as a probe, is now enabling chromosomes and genomes to be located not only at metaphase but throughout the cell cycle, Because probes and detection reagents might n o t penetrate uniformly throughout the interphase nucleus, initial investigations required reconstructions of probed consecutive sections. In barley x Secale a f r i c a n u m , it was evident that the two sets of seven chromosomes from each parent were spatially separated, not only at metaphase but throughout the cell cycle, at least in cells from near the root meristem [24..]. Some variability in patterns was found between cells, indicating that the chromosome location is maintained, or perhaps reinforcecl, by an active mechanism, but may be influenced by developmental or environmental factors.
At anaphase in barley x S. a f r i c a n u m hybrids, the two parental genomes separate simultaneously. In contrast, in another hybrid, barley x 14. bulbosum, which also shows genome separation throughout the cell cycle, the chromosomes from the H. b u l b o s u m genome lag during division [25]. Thus, organization of the nucleus during division, and spatial locations of whole sets of chromosomes, can be modulated during the cell cycle, but the genetic control of this behaviour is evidenced by the contrasting behaviour in different hybrids.
Meiotic reorganization Examination of locations of single pairs of homologous chromosomes in somatic interphase nuclei confirms the impression gained from metaphase analysis that homologues are not paired during most of the cell cycle [26-.]. In a hybrid plant, metaphase analysis indicates that homologous chromosomes, which may pair during the early ~tages of meiosis, are still apart at the last somatic metaphase before meiosis [27"]. Thus, a major physical reorganization of the nucleus occurs during the extended last pre-meiotic interphase and before pachytene of meiosis. In the human [28], yeast [29..], and some cereals (T. Schwarzacher, unpublished data), we can begin to follow whole chromosomes or chromosome arms during the pre-meiotic interphase and meiotic prophase by in situ hybridization of probes that paint whole chromosomes or chromosome arms. In the next few years, we can look forward to a detailed analysis of when chromosome pairing occurs, where it is initiated along the chromosomes, and when and how recombination takes place. Perhaps we shall also find the exact role of synaptonemal complex: evidence from yeast mutants indicates that the structure is independent of recombination [30]. In the cereals, the physical clustering of genes near the ends of chromosomes is not evident on genetic maps
because most recombination occurs in this region. Do chromosomes condense differentially during meiosis to reflect the recombination sites? At least by pachytene, markers of heterochromatin and rDNA seem to be in the same positions along chromosome arms as at mitosis [31"]. It is noteworthy that the sizes of rDNA sites, as measured by in situ hybridization, to occupy much more of the chromosome length at mitosis than at meiosis; the meiotic measurements correlate closely with the proportion of the genome that is rDNA.
The nucleolus The nucleolus is the most conspicuous feature of nuclear architecture in the light microscope. The remarkable controversy over its structure, and, in particular, where the transcribing genes lie, continues. Jordan [32] has presented seven models, each supported by some evidence concerning the internal structure, organization and activity of the body. High-resolution in situ hybridization of rDNA probes to electron microscope preparations has n o w enabled re-interpretation of light microscope data in both human [33] and plant nucleoli [34.]. The similarity between the morphologically distinct nucleoli is striking, with both showing active, diffuse genes near the dense fibrillar component, although there are substantial differences in the localization of condensed, unexpressed genes even in species as closely related as wheat and rye [34°]. Unmethylated cytosine residues at specific sites within the rDNA repeat unit correlate with gene expression and perturbation of chromatin structure [35"]. A cytological stud), of the nucleolus is indeed close to being associated with a molecular investigation of gene structure.
Conclusions Molecular cytogenetic studies using in situ hybridization at light and electron microscope levels are now allow. ing the structure of the interphase nucleus to be directly probed. For the first time we can localize a particular DNA sequence within any nucleus at any developmental stage. As a result, relationships between nuclear architecture and function, evolution, genetics and molecular biology are rapidly emerging. Comparative studies, covering a range in nuclear DNA contents and using hybrids, are enabling us to describe general features of nuclear and chromosomal architecture in plants.
Acknowledgements I am grateful to Trude Schwarzacher, Andrew keitch, Kesara Anamthawat-J6nsson, Ilia Leitch, Gill Harrison and Shi Min for their enormous contributions to my research program. I thank them and Mikael Roose for helpful comments, and many other authors for
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Genomes and evolution pre-prints. I thank BP Venture Research and AFRC for supporting my research programme.
to become methylated and inactivated, and gene suppression is correlated closely with methylation. Stable expression of inserted genes will require the absence of modifier sequences causing such suppression. 10,
OLIVERSG, VAN DER AART QJM, AGOSTON1-CARBONEML, AIGLE M, ALBERGHINA L, ALEXANDRAKI D, ANTOINE G, ANWAR R, BALLESTAJPG, BENIT P, ET AL:The Complete DNA Sequence of Yeast C h r o m o s o m e IIi. Nature 1992, 357:38-46. The first complete sequence of a eukaryotic chromosome. Many, ORFs of unknown function were identified. The comparison of the physical (sequence) map with the genetic map shows regions of increased and decreased meiotic recombination. .o
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest •, of outstanding interest BENNETTMD, SMITH JB: Nuclear DNA A m o u n t s in An. giosperms. Phil Trans R Soc Lond [Biol] 1991, 334:309--345. This is a compendium of genome sizes, adding to three previous collected lists by the same authors, and increasing the number of plant species where DNA amounts have been measured to over 1500 (of the 250 000 angiospeml species known). It is of wide use to cell and molecular biologists (as well as ecologists and taxonomists) who are interested in genome analysis and tile consequences of absolute nuclear genome size.
11.
1.
2. •
GAmP, ArrH DW, HARmNS KR, KNAPP S: Systemic Endopolyploidy in Arabidopsis thaliana. Plant Ph3~iol 1991, 96:985-989. Endoreduplication is found in all somatic tissues of tile species except for the intlorescences. The concept of a need for a minimal mass of nuclear DNA to maintain specific regulator3, and functional states is extensively discussed. 3. •
MALUSZYNSKA J, HESLOP-HARRISONJS: Localization of Tandemly Repeated DNA Sequences in Arabidopsis thaliana. Plant J 1991, 1:159-166. Fluorescent staining methods enable the vet3 , small chromosomes of Arabidopsis to be easily studied. In situ hybridization is used to map the rDNA sequences (8% of the genome) to two chromosome pairs. A centromeric repeat sequence, about 1.5% of the genome, is mapped to all five chromosome pairs. 4.
ANATOFIAWAT-JONSSONK, HESLOP-HARRLSONJS: Centromeres, Telomeres and Chromatin in t h e Interphase Nucleus of Cereals. Caryologia 1990, 43:205-213.
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WATERBORGJH: Existence of T w o Histone H3 Variants in Dicotyledonous Plants and Correlation Between their Acetylation and Plant G e n o m e Size. Plant Mol Biol 1992, 18:181-187.
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WANNERG, FORMANEK H, MARTIN R, HERRbtANNRG: High Res-
•
olution Scanning Electron Microscopy of Plant Chromosomes. Cbromosoma 1991, 100:103-109. This paper reports field emission scanning electron micrographs of spread barley chromosomes, imaging structures down to 10nm. A detailed technique is presented, and the method has great potential for anab/sis of chromosome condensation and decondensation. 7. •,
SLATI'ERRE, DUPREE P, GRAYJC: A Scaffold-associated DNA Region is Located D o w n s t r e a m o f the Pea Plastocyanin Gene. Plant Cell 1991, 3:1239-1250. Not only, detailed and careful work describing the first isolation and sequencing of a SAR DNA region from plants, but an excellent introduction that puts the research findings into the context of nuclear architecture and the related animal work. 8.
HALL G, ALLENGC, LOER DS, THOMPSON WF: Nuclear Scaffolds and Scaffold-attachment Regions in Higher Plants. Proc Natl Acad Sci USA 1991, 88:9320-9324.
MATZKE /VIA, MATZKE AJM: Differential Inactivation and Methylation of a T r a n s g e n e in Plants by T w o Suppressor Loci Containing Homologous Sequences. Plant Mol Biol 1991, 16:821-830. In doubly transformed tobacco plants, an inserted marker gene can be unexpectedly inactivated when a second and different gene is inserted in an unlinked site. Here, gene constructs are used that give complete or partial suppression of the first locus in transformed plants. The second transforming DNA can, depending on the construct, cause the first gene
Gva~ E, SOMERVIH.E C: Construction and Characterization of a Yeast Artificial C h r o m o s o m e Library of Arabtdopsis w h i c h is Suitable for C h r o m o s o m e Walking. Mol Gen Genet 1991, 226:484--490.
SCHMIDT R, DEAN C: Physical Mapping of t h e Arabidopsis thaliana Genome. In Genome Analysis: Strategies for Pto,sical Mapping. Edited by Davies KR. Cold Spring Harbor: Cold Spring Harbor Press; 1992, in press. A description of the strategies for, and progress in, generating physical maps from c h r o m o s o m e - ~ l k i n g experiments ,and making an overlapping clone library in this model plant species. 12. ..
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GUSTAFSONJP, DILLEJE: T h e Chromosomal Location of Oryza Sativa Recombination Linkage Groups 1,2. Proc Natl Acad Sci USA 1992, 89:8646-8650.
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LEITCH IJ, HESLOP-HARRISON JS: Physical Mapping of the 18S-5.8S--26S rRNA G e n e s in Barley by in Situ Hybridization. Genome 1992, in press. High-resolution in situ hybridization is able to map five sites of rRNA genes in barley, compared to the two mapped by other methods. There are four sites of 5S rRNA genes, rather than the one mapped previously. The cx-amylase gene is distal on c h r o m o s o m e 5H. Comparison of the physical locations of these genes with the genetic locations shows that most recombination occurs at tile ends of chromosomes. •
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HESLOP-HARRISONJS: T h e Molecular Cytogenetics of Plants. J Cell Sci 1991, 100:15-21.
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DOONERHK: Genetic Fine Structure of t h e Bronze Locus in Maize. Genetics 1986, 113:1021-1036.
MOOREG, CHEUNG W, FOOTE T, GALE M, KOEBNER R, LEITCH AR, LEITCH lJ, MONEY T, STANCOMBE P, YANO M, FLAVELLPc Key Features of Cereal G e n o m e Organisation as Revealed by the Use of Cytosine Methylation-sensitive Restriction Endonucleases. Genomics 1992, in press. An extensive paper that brings together molecular, genetical and cytological experiments to begin to show how large genomes are organized, where repetitive DNA is localized, and how different sequence families are spatially related to each other along the chromosomes. The data provide a basis for targeted gene isolation and manipulation of large genomes. 17. •e
18.
CHEUNGWY, MOORE G, MONEY TA, GALE MD: Hpall Library Indicates 'Methylation-free Islands' in W h e a t and Barley. Theor Appl Genet 1992, 84:739-746.
19. .
LINDE-LAURSENI, VON BOTHMER R: Barley C h r o m o s o m e 6 Rerained Specifically in Hypoploid Hybrids of a C h r o m o s o m e eUminating Interspecific Cross. Barley Genet 1991, 6:77-79. Loss of chromosomes occurs during cell division. This reports the preferential retention of o n e c h r o m o s o m e in hybrids, indicaUng that mechanisms ensure segregation of particular chromosomes. 20.
FINCHRA, BENNETF MD: T h e M e c h a n i s m of Somatic Chromos o m e Elimination in Hordeum. Kew Chromosome Conference. Edited by Brandham PE, Bennett MD. London: Allan & Unwin; 1983, 2:147-154.
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HESLOP-HARRISONJS, BENNETT MD: Nuclear Architecture in Plants. Trends Genet 1990, 6:401-405.
22.
LINDE-LAURSENI, JENSEN J: G e n o m e and C h r o m o s o m e Disposition at Somatic Metaphase in a H o r d e u m x Psathyrostachys Hybrid. Heredity 1991, 66:203-210.
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Nuclear architecture in plants 23. •
BENNETI"ST, BENNETI" MD: Spatial Separation of Ancestral G e n o m e s in the Wild Grass M i l i u m m o n t i a n u m Pari. Ann Bot 1992, 70:111-118. M. montianum is a wild allotetraploid grass. In both mitotic and meiotic nuclei, the ancestral genomes tended to lie apart in spread metaphase preparations. It is suggested that the nuclear organization is under genetic control and may facilitate independent behaviour of the genomes in the polyploid, leading to speciation. 24. ••
LEITCH AR, SCHWARZACHERT, MOSGOLLER W, BENNETT MD, HESLOP-HARRISONJS: Parental G e n o m e s are Separated T h r o u g h o u t the Cell Cycle in a Plant Hybrid. Cbromosoma 1991, 101:206-213. In situ hybridization is used to localize whole genomes in a sexual hybrid plant (barley x S. africanam, both n = 7) on both spread and sectioned preparations. Reconstructions show that the one chromosome set is located around the nucleolus, while the other occupies one, two or three domains in the peripheral region of the nucleus. 25.
ANAMTHAWAT-JON~SON K, SCHWARZACHERT, HESLOP-HARRISONJS: Behavior of Parental G e n o m e s in H o r d e u m vulgate x H. bulbosum. J Hered 1992, in press.
26. ••
SCHWARZACHER T, ANAMTHAWAT-JONSSONK, HARRISON GE, ISt~t AK&IR,JIA JZ, KING IP, LEITCH AR, MILLER TE, READER SM, ROGERS WJ, bY/"AL: G e n o m i c in Situ Hybridization to Identify Alien C h r o m o s o m e s and C h r o m o s o m e Segments in Wheat. 77Jeor Appl Genet 1992, 84:778-786. Total genomic DNA can be used as a probe for in situ hybridization to identify chromosomes and chromosome segments introduced from many different ('alien') species into wheat. The method also shows the locations of the alien chromosomes at interphase; in diploid lines, the alien chromosomes are not associated but lie in different domains. 27.
SCtTWARZACHERT, HESLOP.HARRISON JS, ANAMTHAWAT-JONSSON K, FINCH RA, BENNE'VI" MD: Parental G e n o m e Separation in Reconstructions of Somatic and Premeiotic Metaphases of H o r d e u m vulgare x H. bulbosum. J Cell Sci 1992, 101:13-24. Serial section reconstructions enable analysis of the three-dinaensional location and identification of each chromosome. The parental genomes are apart in root-tip cells and pollen mother cells at the last mitotic division before meiosis. The authors speculate that the nucleus is reorganized physically to align homoeologous or, in species, homologous, chromosomes, after this mitosis, but before meiosis.
•
28.
29. •,
GOLD/~L,XNASH, HULTEN MA: C h r o m o s o m e in Situ Suppression Hybridization in H u m a n Male Meiosis. J Med Genet 1992, 29:98-102.
SCHERTHANH, LOIDL J, SCHUSTER T, SCHWEIZER D: Meiotic C h r o m o s o m e Condensation and Pairing in S. cerevisiae Studied by C h r o m o s o m e Painting. Chromosoma 1992, 101:590-595. Movements of homologous c h r o m o s o m e segments were followed during early meiotic prophase by in situ hybridization of pools of DNA
Heslop-Harrison
clones. The chromatin was reported to condense, and homologous segments align, before synaptonemal complex formation, which has major implications for models of chromosome recognition, recombination and pairing. 30.
HABERJE, LEUNGW-Y, BORTS R.H, LICHTEN M: The Frequency of Meiotic Recombination in Yeast is I n d e p e n d e n t of t h e N u m b e r and Position of Homologous Donor Sequences: Implications for C h r o m o s o m e Pairing. Proc Natl Acad Sci USA 1991, 88:1120-1124.
AtBINI SM, SCHWAP,ZACHER T: I n Sign Localization of T w o Repetitive DNA Sequences to Surface-spread Pachytene C h r o m o s o m e s of Rye. Genome 1992, 35:551-559. Meiotic prophase nuclei were surface-spread and used for in situ hybridization with the rDNA sequence and a heterochromatin sequence. The DNA associated with the bivalent axes (and synaptonemal complex) was stained strongly with DNA fluorochromes and probe hybridization occurred along the taxes. Tile method will be valuable for high-resolution physical mapping of DNA sequences and investigation of the locations of defined DNA sequences involved in pairing and recombination. 31. •
32.
JORDANEG: Interpreting Nucleolar Structure: W h e r e are the Transcribing Genes? J Cell Sci 1991, 98:437-442.
33.
WACHTLER F, MOSGOLLERW, SCHWARZ.AGHERHG: Electron Microscopic in Situ Hybridization and Autoradiography: Localization and Transcription of rDNA in H u m a n Lymphocyte Nucleoli. E.xp Cell Res 1990, 187:346-348.
34. •
I.ErrCHAR, MOSGOLLERW, SHI M, HESLOP-HARRISONJS: Differ. e n t Patterns of rDNA Organization at lnterphase in Nuclei of W h e a t and Rye. J Cell Sci 1992, 101:751-757. In situ hybridization, using light and electron microscopy is used to 1o. cate rRNA genes within interphase nucleoli. In wheat, condensed rDNA foci are visible within the nucleolus, with fine threads between them called fragmented decondensation. Only rye has the diffuse sites, which are likely to represent active rRNA genes. Thus, the organization of gene expression is fundamentally different in the two related species. 35. .•
SARDANAR, FLAVELLRB, O'DELL M: Correlation b e t w e e n t h e Size of the Intergenic Regulatory Region, t h e Status of Cytosine Methylation of rRaNA G e n e s and Nucleolar Expression in Wheat. Mol Gen Genet 1992, in press. Relative nucleolus organizer (rDNA site) activity, is correlated with the number of repeats of a 135 bp sequence upstream of the gene promoter. Active loci include more gene repeat units where there are unmethylated cytosines at specific sites in the promoter and in the 135 bp repeat, and in which chromatin structure is perhaps altered.
JS Heslop-Harrison, Department of Cell Biology, John lnnes Centre for Plant Science Research, Colney Lane, Norwich NR4 7UJ, UK.
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