Partial Publication List Trude Schwarzacher

1: Ann Bot (Lond). 2007 Aug 31; [Epub ahead of print] Related Articles, LinkOut
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Domestication, Genomics and the Future for Banana.

Heslop-Harrison JS, Schwarzacher T.

Department of Biology, University of Leicester, Leicester LE1 7RH, UK.

Background Cultivated bananas and plantains are giant herbaceous plants within the genus Musa. They are both sterile and parthenocarpic so the fruit develops without seed. The cultivated hybrids and species are mostly triploid (2n = 3x = 33; a few are diploid or tetraploid), and most have been propagated from mutants found in the wild. With a production of 100 million tons annually, banana is a staple food across the Asian, African and American tropics, with the 15 % that is exported being important to many economies. Scope There are well over a thousand domesticated Musa cultivars and their genetic diversity is high, indicating multiple origins from different wild hybrids between two principle ancestral species. However, the difficulty of genetics and sterility of the crop has meant that the development of new varieties through hybridization, mutation or transformation was not very successful in the 20th century. Knowledge of structural and functional genomics and genes, reproductive physiology, cytogenetics, and comparative genomics with rice, Arabidopsis and other model species has increased our understanding of Musa and its diversity enormously. Conclusions There are major challenges to banana production from virulent diseases, abiotic stresses and new demands for sustainability, quality, transport and yield. Within the genepool of cultivars and wild species there are genetic resistances to many stresses. Genomic approaches are now rapidly advancing in Musa and have the prospect of helping enable banana to maintain and increase its importance as a staple food and cash crop through integration of genetical, evolutionary and structural data, allowing targeted breeding, transformation and efficient use of Musa biodiversity in the future.

PMID: 17766312 [PubMed - as supplied by publisher]

2: Gene. 2007 Jul 19; [Epub ahead of print] Related Articles, LinkOut
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Repetitive DNA, molecular cytogenetics and genome organization in the King scallop (Pecten maximus).

Biscotti MA, Canapa A, Olmo E, Barucca M, Teo CH, Schwarzacher T, Dennerlein S, Richter R, Heslop-Harrison JS.

Universit√  Politecnica delle Marche, Italy.

We studied the structure, organization and relationship of repetitive DNA sequences in the genome of the scallop, Pecten maximus, a bivalve that is important both commercially and in marine ecology. Recombinant DNA libraries were constructed after partial digestion of genomic DNA from scallop with PstI and ApaI restriction enzymes. Clones containing repetitive DNA were selected by hybridisation to labelled DNA from scallop, oyster and mussel; colonies showing strong hybridisation only to scallop were selected for analysis and sequencing. Six non-homologous tandemly repeated sequences were identified in the sequences, and Southern hybridisation with all repeat families to genomic DNA digests showed characteristic ladders of hybridised bands. Three families had monomer lengths around 40 bp while three had repeats characteristic of the length wrapping around one (170 bp), or two (326 bp) nucleosomes. In situ hybridisation to interphase nuclei showed each family had characteristic numbers of clusters indicating contrasting arrangements. Two of the repeats had unusual repetitions of bases within their sequence, which may relate to the nature of microsatellites reported in bivalves. The study of these rapidly evolving sequences is valuable to understand an important source of genomic diversity, has the potential to provide useful markers for population studies and gives a route to identify mechanisms of DNA sequence evolution.

PMID: 17706376 [PubMed - as supplied by publisher]

3: Anal Bioanal Chem. 2007 Aug 11; [Epub ahead of print] Related Articles, LinkOut
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DNA aptamers against the MUC1 tumour marker: design of aptamer-antibody sandwich ELISA for the early diagnosis of epithelial tumours.

Ferreira CS, Papamichael K, Guilbault G, Schwarzacher T, Gariepy J, Missailidis S.

Chemistry Department, The Open University, Walton Hall, Milton Keynes, MK7 6AA, UK, s.missailidis(a)open.ac.uk.

Aptamers are functional molecules able to bind tightly and selectively to disease markers, offering great potential for applications in disease diagnosis and therapy. MUC1 is a well-known tumour marker present in epithelial malignancies and is used in immunotherapeutic and diagnostic approaches. We report the selection of DNA aptamers that bind with high affinity and selectivity an MUC1 recombinant protein containing five repeats of the variable tandem repeat region. Aptamers were selected using the SELEX methodology from an initial library containing a 25-base-long variable region for their ability to bind to the unglycosylated form of the MUC1 protein. After ten rounds of in vitro selection and amplification, more than 90% of the pool of sequences consisted of target-binding molecules, which were cloned, sequenced and found to share no sequence consensus. The binding properties of these aptamers were quantified using ELISA and surface plasmon resonance. The lead aptamer sequence was subsequently used in the design of an aptamer-antibody hybrid sandwich ELISA for the identification and quantification of MUC1 in buffered solutions. Following optimisation of the operating conditions, the resulting enzyme immunoassay displayed an EC(50) value of 25 mug/ml, a detection limit of 1 mug/ml and a linear range between 8 and 100 mug/ml for the MUC1 five tandem repeat analyte. In addition, recovery studies performed in buffer conditions resulted in averaged recoveries between 98.2 and 101.7% for all spiked samples, demonstrating the usability of the aptamer as a receptor in microtitre-based assays. Our results aim towards the formation of new diagnostic assays against this tumour marker for the early diagnosis of primary or metastatic disease in breast, bladder and other epithelial tumours. Figure An aptamer-antibody two-dimentional immunoassay for MUC1.

PMID: 17694298 [PubMed - as supplied by publisher]

4: BMC Plant Biol. 2007 May 21;7:24. Related Articles, Cited Articles, CoreNucleotide, Gene, Taxonomy via GenBank, UniGene, Nucleotide, Protein, Free in PMC, LinkOut
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Endogenous pararetroviral sequences in tomato (Solanum lycopersicum) and related species.

Staginnus C, Gregor W, Mette MF, Teo CH, Borroto-Fern√°ndez EG, Machado ML, Matzke M, Schwarzacher T.

Gregor Mendel Institute of Plant Molecular Biology (GMI), Wien, Austria. christina.staginnus(a)gmi.oeaw.ac.at

BACKGROUND: Endogenous pararetroviral sequences (EPRVs) are a recently discovered class of repetitive sequences that is broadly distributed in the plant kingdom. The potential contribution of EPRVs to plant pathogenicity or, conversely, to virus resistance is just beginning to be explored. Some members of the family Solanaceae are particularly rich in EPRVs. In previous work, EPRVs have been characterized molecularly in various species of Nicotiana including N.tabacum (tobacco) and Solanum tuberosum (potato). Here we describe a family of EPRVs in cultivated tomato (Solanum lycopersicum L.) and a wild relative (S.habrochaites). RESULTS: Molecular cloning and DNA sequence analysis revealed that tomato EPRVs (named LycEPRVs) are most closely related to those in tobacco. The sequence similarity of LycEPRVs in S.lycopersicum and S.habrochaites indicates they are potentially derived from the same pararetrovirus. DNA blot analysis revealed a similar genomic organization in the two species, but also some independent excision or insertion events after species separation, or flanking sequence divergence. LycEPRVs share with the tobacco elements a disrupted genomic structure and frequent association with retrotransposons. Fluorescence in situ hybridization revealed that copies of LycEPRV are dispersed on all chromosomes in predominantly heterochromatic regions. Methylation of LycEPRVs was detected in CHG and asymmetric CHH nucleotide groups. Although normally quiescent EPRVs can be reactivated and produce symptoms of infection in some Nicotiana interspecific hybrids, a similar pathogenicity of LycEPRVs could not be demonstrated in Solanum L. section Lycopersicon [Mill.] hybrids. Even in healthy plants, however, transcripts derived from multiple LycEPRV loci and short RNAs complementary to LycEPRVs were detected and were elevated upon infection with heterologous viruses encoding suppressors of PTGS. CONCLUSION: The analysis of LycEPRVs provides further evidence for the extensive invasion of pararetroviral sequences into the genomes of solanaceous plants. The detection of asymmetric CHH methylation and short RNAs, which are hallmarks of RNAi in plants, suggests that LycEPRVs are controlled by an RNA-mediated silencing mechanism.

5: Gene. 2006 Mar 1;368:61-71. Epub 2005 Dec 1. Related Articles, CoreNucleotide, Nucleotide, LinkOut
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The origin and evolution of the variability in a Y-specific satellite-DNA of Rumex acetosa and its relatives.

Navajas-Perez R, Schwarzacher T, de la Herran R, Ruiz Rejon C, Ruiz Rejo≥n M, Garrido-Ramos MA.

Departamento de Genetica, Facultad de Ciencias, Universidad de Granada, 18071 Granada, Spain.

In this paper, we analyze a satellite-DNA family, the RAYSI family, which is specific of the Y chromosomes of Rumex acetosa, a dioecious plant species with a multiple sex-chromosome system in which the females are XX and the males are XY(1)Y(2). Here, we demonstrate that this satellite DNA is common to other relatives of R. acetosa, including Rumex papillaris, Rumex intermedius, Rumex thyrsoides and Rumex tuberosus that are also dioecious species with a multiple system of sex chromosomes. This satellite-DNA family is absent from the genomes of other dioecious Rumex species having an XX/XY sex-chromosome system. Our data confirm recent molecular phylogenies that support a unique origin for all dioecious species of Rumex and two separate lineages for species with single or complex sex-chromosome systems. Our data also support an accelerated degeneration of Y-chromosome in XX/XY(1)Y(2) species by the accumulation of satellite-DNA sequences. On the other hand, the particular non-recombining nature of the Y chromosomes of R. acetosa and their closest relatives lead to a particular mode of evolution of RAYSI sequences. Thus, mechanisms leading to the suppression of recombination between the Y chromosomes reduced the rate of concerted evolution and gave rise to the apparition of different RAYSI subfamilies. Thus, R. acetosa and R. intermedius have two subfamilies (the RAYSI-S and RAYSI-J subfamilies and the INT-A and INT-B subfamilies, respectively), while R. papillaris only has one, the RAYSI-J subfamily. The RAYSI-S and RAYSI-J subfamilies of R. acetosa differ in 83 fixed diagnostic sites and several diagnostic deletions while the INT-A and the INT-B of R. intermedius differ in 27 fixed diagnostic sites. Pairwise comparisons between RAYSI-S and RAYSI-J sequences or between INT-A and INT-B sequences revealed these sites to be shared mutations detectable in repeats of the same variant in same positions. Evolutionary comparisons suggest that the subfamily RAYSI-J has appeared in the common ancestor of R. acetosa and R. papillaris, in which RAYSI-J has replaced totally (R. papillaris) or almost totally the ancestral sequence (R. acetosa). This scenario assumes that RAYSI-S sequences should be considered ancestral sequences and that a secondary event of subfamily subdivision should be occurring in R. intermedius, with their RAYSI subfamilies more closely related to one another than with other RAYSI sequences. Our analysis suggests that the different subfamilies diverged by a gradual and cohesive way probably mediated by sister-chromatid interchanges while their expansion or contraction in number might be explained by alternating cycles of sudden mechanisms of amplification or elimination.


PMID: 16324803 [PubMed - indexed for MEDLINE]

6: Cytogenet Genome Res. 2005;109(1-3):34-42. Related Articles, CoreNucleotide, Nucleotide, LinkOut
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Diversity of a major repetitive DNA sequence in diploid and polyploid Triticeae.

Contento A, Heslop-Harrison JS, Schwarzacher T.

Department of Biology, University of Leicester, Leicester, UK.

About 90 members of a major tandemly repeated DNA sequence family originally described in rye as pSc119.2 have been isolated from 11 diploid and polyploid Triticeae species using primers from along the length of the sequence for PCR amplification. Alignment and similarity analysis showed that the 120-bp repeat unit family is diverse with single nucleotide changes and few insertions and deletions occurring throughout the sequence, with no characteristic genome or species-specific variants having developed during evolution of the extant genomes. Fluorescent in situ hybridization showed that each of the large blocks of the repeat at chromosomal sites harboured many variants of the 120-bp repeat. There were substantial copy number differences between genomes, with abundant sub-terminal sites in rye, interstitial sites in the B genome of wheat, and relatively few sites in the A and D genome. We conclude that sequence homogenization events have not been operative in this repeat and that the common ancestor of the Triticeae tribe had multiple sequences of the 120-bp repeat with a range of variation not unlike that seen within and between species today. This diversity has been maintained when sites are moved within the genome and in all species since their divergence within the Triticeae. Copyright 2005 S. Karger AG, Basel.

PMID: 15753556 [PubMed - indexed for MEDLINE]

7: Ann Bot (Lond). 2004 Nov;94(5):699-705. Epub 2004 Sep 8. Related Articles, LinkOut
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High levels of genetic diversity throughout the range of the Portuguese wheat landrace 'Barbela'.

Ribeiro-Carvalho C, Guedes-Pinto H, Igrejas G, Stephenson P, Schwarzacher T, Heslop-Harrison JS.

Department of Biology, University of Leicester, Leicester LE1 7RH, UK. ccarvalh(a)utad.pt

BACKGROUND AND AIMS: Landrace populations represent an important intra-crop reservoir of biodiversity and source of novel gene alleles for use in breeding programmes. Here the aim was to measure the diversity of a wheat landrace, 'Barbela', from the north of Portugal. METHODS: DNA was extracted from 59 accessions of Barbela collected across its geographical range. Diversity was measured by microsatellite length polymorphisms using 27 primer pairs amplifying 34 polymorphic microsatellite loci. KEY RESULTS: High levels of polymorphism were found, with an average polymorphism information content of 0.52; an average of 4.77 alleles (range 2-11) were present at each locus, and half of these loci showed an additional allele in the reference variety 'Chinese Spring'. CONCLUSIONS: 'Barbela' is maintained from seeds collected by farmers, but it maintains high allelic variation, and no groupings of accessions were detected when analysed by geographical region, farm or climate, indicating that the wheat landrace is a homogeneous entity. The diversity within the farmer-maintained landrace demonstrates the importance of characterization and maintenance of landrace collections before valuable genetic combinations are lost as uniform commercial crops are introduced.

PMID: 15355867 [PubMed - indexed for MEDLINE]

8: Genome. 2003 Dec;46(6):953-62. Related Articles, Substance (MeSH Keyword), Cited in PMC, LinkOut
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DNA, chromosomes, and in situ hybridization.

Schwarzacher T.

Department of Biology, University of Leicester, UK. ts32(a)le.ac.uk

In situ hybridization is a powerful and unique technique that correlates molecular information of a DNA sequence with its physical location along chromosomes and genomes. It thus provides valuable information about physical map position of sequences and often is the only means to determine abundance and distribution of repetitive sequences making up the majority of most genomes. Repeated DNA sequences, composed of units of a few to a thousand base pairs in size, occur in blocks (tandem or satellite repeats) or are dispersed (including transposable elements) throughout the genome. They are often the most variable components of a genome, often being species and, occasionally, chromosome specific. Their variability arises through amplification, diversification and dispersion, as well as homogenization and loss; there is a remarkable correlation of molecular sequence features with chromosomal organization including the length of repeat units, their higher order structures, chromosomal locations, and dispersion mechanisms. Our understanding of the structure, function, organization, and evolution of genomes and their evolving repetitive components enabled many new cytogenetic applications to both medicine and agriculture, particularly in diagnosis and plant breeding.

PMID: 14663512 [PubMed - indexed for MEDLINE]

9: EMBO J. 2003 Sep 15;22(18):4836-45. Related Articles, Cited Articles, CoreNucleotide, Taxonomy via GenBank, Nucleotide, Protein, Free in PMC, Cited in PMC, LinkOut
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Induction of infectious petunia vein clearing (pararetro) virus from endogenous provirus in petunia.

Richert-Poggeler KR, Noreen F, Schwarzacher T, Harper G, Hohn T.

Friedrich Miescher Institute, PO Box 2543, CH-4002 Basel, Switzerland. richert(a)fmi.ch

Infection by an endogenous pararetrovirus using forms of both episomal and chromosomal origin has been demonstrated and characterized, together with evidence that petunia vein clearing virus (PVCV) is a constituent of the Petunia hybrida genome. Our findings allow comparative and direct analysis of horizontally and vertically transmitted virus forms and demonstrate their infectivity using biolistic transformation of a provirus-free petunia species. Some integrants within the genome of P.hybrida are arranged in tandem, allowing direct release of virus by transcription. In addition to known inducers of endogenous pararetroviruses, such as genome hybridization, tissue culture and abiotic stresses, we observed activation of PVCV after wounding. Our data also support the hypothesis that the host plant uses DNA methylation to control the endogenous pararetrovirus.
PMID: 12970195 [PubMed - indexed for MEDLINE]

10: Chromosome Res. 2003;11(3):241-53. Related Articles, Cited in PMC, LinkOut
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Tandemly repeated DNA sequences and centromeric chromosomal regions of Arabidopsis species.

Heslop-Harrison JS, Brandes A, Schwarzacher T.

CREST Project, Department of Biology, University of Leicester, Leicester LE1 7RH, UK. phh4(a)le.ac.uk

Despite their common function, centromeric DNA sequences are not conserved between organisms. Most centromeres of animals and plants so far investigated have now been shown to consist of large blocks of tandemly repeated satellite sequences that are embedded in recombination-deficient heterochromatic regions. This central domain of satellite sequences that is postulated to mediate spindle attachment is surrounded by pericentromeric sequences incorporating various classes of repetitive sequences often including retroelements. The centromeric satellite DNA sequences are amongst the most rapidly evolving sequences and pose some fundamental problems of maintaining function. In this overview, we will discuss work on centromeric repetitive sequences in Arabidopsis thaliana and its relatives, and highlight some of the common features that are emerging when analysing closely related species.

PMID: 12769291 [PubMed - indexed for MEDLINE]

11: J Exp Bot. 2003 Jan;54(380):11-23. Related Articles, Cited in PMC, LinkOut
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Meiosis, recombination and chromosomes: a review of gene isolation and fluorescent in situ hybridization data in plants.

Schwarzacher T.

Department of Biology, University of Leicester, University Road, Leicester LE1 7RH, UK. TS32(a)le.ac.uk

Evidence is now increasing that many functions and processes of meiotic genes are similar in yeast and higher eukaryotes. However, there are significant differences and, most notably, yeast has considerably higher recombination frequencies than higher eukaryotes, different cross-over interference and possibly more than one pathway for recombination, one late and one early. Other significant events are the timing of double-strand breaks (induced by Spo11) that could be either cause or consequence of homologous chromosome synapsis and SC formation depending on the organisms, yeast plants and mammals versus Drosophila melanogaster and Caenorhabditis elegans. Many plant homologues and heterologues to meiotic genes of yeast and other organisms have now been isolated, in particular in Arabidopsis thaliana, showing that overall recombination genes are very conserved while synaptonemal complex and cohesion proteins are not. In addition to the importance of unravelling the meiotic processes by gene discovery, this review discusses the significance of chromatin packaging, genome organization, and distribution of specific repeated DNA sequences for homologous chromosome cognition and pairing, and the distribution of recombination events along the chromosomes.

PMID: 12456751 [PubMed - indexed for MEDLINE]

12: J Biochem Mol Biol Biophys. 2002 Jun;6(3):193-201. Related Articles, CoreNucleotide, Substance (MeSH Keyword), Taxonomy via GenBank, Nucleotide, Protein, PopSet, LinkOut
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The cloning of Ty1-copia-like retrotransposons from 10 varieties of banana (Musa Sp.).

Teo CH, Tan SH, Othman YR, Schwarzacher T.

Department of Biotechnology, Faculty of Food Science and Biotechnology, University Putra Malaysia, 43400 Serdang, Selangor, Malaysia.

Ty1-copia-like retrotransposons have been identified and investigated in several plant species. Here, the internal region of the reverse transcriptase (RT) gene of Ty1-copia-like retrotransposons was amplified by PCR from total genomic DNA of 10 varieties of banana. Two to four clones from each variety were sequenced. Extreme heterogeneity in the sequences of Ty1-copia-like retrotransposons from all the varieties was revealed following sequence analysis of the reverse transcriptase (RT) fragments. The size of the individual RT gene fragments varied between 213 and 309 bp. Southern blots of genomic DNA digested from Musa acuminata and other banana varieties probed with W8 clone from M. acuminata and A4 clone from Pisang Abu Nipah showed similar strong, multiple restriction fragments together with other faint hybridization band patterns with variable intensities indicating the presence of many copies of the Ty1-copia-like retrotransposons in the genomes. There was no correlation between retroelement sequence and the banana species (with A or B genomes) from which it arose, suggesting that the probes are not useful for tracking genomes through breeding populations.

PMID: 12186754 [PubMed - indexed for MEDLINE]

13: Heredity. 2002 May;88(5):349-55. Related Articles, LinkOut
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Characterisation of mildew resistant wheat-rye substitution lines and identification of an inverted chromosome by fluorescent in situ hybridisation.

Forsström PO, Merker A, Schwarzacher T.

Department of Crop Science, Swedish University of Agricultural Sciences, SE-230 53 Alnarp, Sweden. Per-Olov.Forsstrom(a)vv.slu.se

Seven different mildew resistant wheat lines derived from crosses between triticale and bread wheat were examined by molecular cytogenetics and chromosome C-banding in order to determine their chromosomal composition. Genomic in situ hybridisation (GISH) showed the presence of rye germplasm in all the lines and identified three substitution lines, three double substitution lines and one addition-substitution line. C-banding identified rye chromosomes 1R and 4R in the addition-substitution line, rye chromosomes 1R and 6R in two substitution lines and 1R and 2R in the third line, and rye chromosome 1R in the three substitution lines. Two of the latter lines (7-102 and 7-169) contained a modified form of the chromosome; fluorescent in situ hybridisation (FISH) using five different repetitive DNA-probes showed a pericentric inversion of 1R in both lines. The breakpoints of the 1R inversion were between (1) the 5S rDNA site and the NOR-region on the satellite of the short arm, and (2) between two AAC(5) sites close to the centromere on the long arm. The role of the rye chromosomes in the mildew resistance, the utilisation of the inverted 1R and the significance of the lines in wheat breeding are discussed.

PMID: 11986870 [PubMed - indexed for MEDLINE]

14: Genome. 2001 Dec;44(6):1122-8. Related Articles, LinkOut
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Introgression of rye chromatin on chromosome 2D in the Portuguese wheat landrace 'Barbela.'.

Ribeiro-Carvalho C, Guedes-Pinto H, Heslop-Harrison JS, Schwarzacher T.

Department of Genetics and Biotechnology, ICETA, University of Tr√°s-os-Montes and Alto Douro, Vila Real, Portugal.

The old Portuguese wheat landrace aggregate known as 'Barbela' shows good productivity under the low-fertility conditions often associated with acid soils. The use of genomic rye DNA, in combination with 45S rDNA and the repetitive sequences dpTal and pScl 19.2 as probes, in two sequential in situ hybridization steps enabled the identification of all chromosomes in the 'Barbela' wheat lines and the detection of the introgression of rye-origin chromatin onto wheat chromosome arm 2DL in two of the lines. Amplification of microsatellite loci using published primer pairs showed that the distal segment of wheat chromosome 2DL, which was involved in the rye translocation, was deleted. The identification and characterization of small recombinant chromosome segments in wheat-rye lines may allow their use in plant breeding programmes. Their presence in farmer-maintained material demonstrates the importance of maintaining, characterizing, and collecting landrace material before valuable genetic combinations are lost as uniform commercial crops are introduced.

PMID: 11768216 [PubMed - indexed for MEDLINE]

15: Genome. 2001 Feb;44(1):104-10. Related Articles, Cited in PMC, LinkOut
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An efficient method for the physical mapping of transgenes in barley using in situ hybridization.

Salvo-Garrido H, Travella S, Schwarzacher T, Harwood WA, Snape JW.

John Innes Centre, Norwich Research Park, UK.

The genetic transformation of crops by particle bombardment and Agrobacterium tumefaciens systems have the potential to complement conventional plant breeding programmes. However, before deployment, transgenic plants need to be characterized in detail, and physical mapping is an integral part of this process. Therefore, it is important to have a highly efficient method for transgene detection by fluorescence in situ hybridization (FISH). This study describes a new approach, which provides efficient control of probe length and labelling, both of which play an important role in in situ hybridization of transgenes. The approach is based on reducing the size of the plasmid prior to labelling by nick translation, rather than using the whole or linearized plasmid, or varying the amounts of DNaseI in the nick translation mixture. This provided much more efficient labelling of the probe, which yielded optimal hybridization. minimal fluorescent background, and accurate physical location of the transgene.

PMID: 11269343 [PubMed - indexed for MEDLINE]

16: Cytogenet Cell Genet. 2000;91(1-4):62-6. Related Articles, LinkOut
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The species and chromosomal distribution of the centromeric alpha-satellite I sequence from sheep in the tribe Caprini and other Bovidae.

Chaves R, Guedes-Pinto H, Heslop-Harrison J, Schwarzacher T.

Department of Biology, University of Leicester, Leicester, UK.

The evolution of chromosomes in species in the family Bovidae includes fusion and fission of chromosome arms (giving different numbers of acrocentric and metacentric chromosomes with a relatively conserved total number of arms) and evolution in both DNA sequence and copy number of the pericentromeric alpha-satellite I repetitive DNA sequence. Here, a probe representing the sheep alpha-satellite I sequence was isolated and hybridized to genomic DNA digests and metaphase chromosomes from various Bovidae species. The probe was highly homologous to the centromeric sequence in all species in the tribe Caprini, including sheep (Ovis aries), goat (Capra hircus) and the aoudad or Barbary sheep (Amnotragus lervia), but showed no detectable hybridization to the alpha-satellite I sequence present in the tribe Bovini and at most very weak to species in the tribes Hippotragini, Alcelaphini or Aepycerotini. The sex chromosomes of sheep, goat and aoudad did not contain detectable alpha-satellite I sequence; in sheep, one of the three metacentric autosomal chromosomes does not carry the sequence, while in aoudad, it is essentially absent in three large autosomal pairs as well as the large metacentric chromosome pair. The satellite probes can be used as robust chromosome and karyotype markers of evolution among tribes and increase the resolution of the evolutionary tree at the base of the Artiodactyla. Copyright 2001 S. Karger AG, Basel

PMID: 11173832 [PubMed - indexed for MEDLINE]

17: Chromosoma. 1998 Dec;107(8):587-94. Related Articles, Cited in PMC, LinkOut
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The chromosomal organization of simple sequence repeats in wheat and rye genomes.

Cuadrado A, Schwarzacher T.

John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK.

The physical distribution of ten simple-sequence repeated DNA motifs (SSRs) was studied on chromosomes of bread wheat, rye and hexaploid triticale. Oligomers with repeated di-, tri- or tetra-nucleotide motifs were used as probes for fluorescence in situ hybridization to root-tip metaphase and anther pachytene chromosomes. All motifs showed dispersed hybridization signals of varying strengths on all chromosomes. In addition, the motifs (AG)12, (CAT)5, (AAG)5, (GCC)5 and, in particular, (GACA)4 hybridized strongly to pericentromeric and multiple intercalary sites on the B genome chromosomes and on chromosome 4A of wheat, giving diagnostic patterns that resembled N-banding. In rye, all chromosomes showed strong hybridization of (GACA)4 at many intercalary sites that did not correspond to any other known banding pattern, but allowed identification of all R genome chromosome arms. Overall, SSR hybridization signals were found in related chromosome positions independently of the motif used and showed remarkably similar distribution patterns in wheat and rye, indicating the special role of SSRs in chromosome organization as a possible ancient genomic component of the tribe Triticeae (Gramineae).

PMID: 9933412 [PubMed - indexed for MEDLINE]

18: Plant Cell. 1999 Jan;11(1):31-42. Related Articles, Cited Articles, Free in PMC, Cited in PMC, LinkOut
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Polymorphisms and genomic organization of repetitive DNA from centromeric regions of Arabidopsis chromosomes.

Heslop-Harrison JS, Murata M, Ogura Y, Schwarzacher T, Motoyoshi F.

Research Institute for Bioresources, Okayama University, Kurashiki 710-0046, Japan. phh4(a)le.ac.uk

A highly abundant repetitive DNA sequence family of Arabidopsis, AtCon, is composed of 178-bp tandemly repeated units and is located at the centromeres of all five chromosome pairs. Analysis of multiple copies of AtCon showed 95% conservation of nucleotides, with some alternative bases, and revealed two boxes, 30 and 24 bp long, that are 99% conserved. Sequences at the 3' end of these boxes showed similarity to yeast CDEI and human CENP-B DNA-protein binding motifs. When oligonucleotides from less conserved regions of AtCon were hybridized in situ and visualized by using primer extension, they were detected on specific chromosomes. When used for polymerase chain reaction with genomic DNA, single primers or primer pairs oriented in the same direction showed negligible amplification, indicating a head-to-tail repeat unit organization. Most primer pairs facing in opposite directions gave several strong bands corresponding to their positions within AtCon. However, consistent with the primer extension results, some primer pairs showed no amplification, indicating that there are chromosome-specific variants of AtCon. The results are significant because they elucidate the organization, mode of amplification, dispersion, and evolution of one of the major repeated sequence families of Arabidopsis. The evidence presented here suggests that AtCon, like human alpha satellites, plays a role in Arabidopsis centromere organization and function.

PMID: 9878630 [PubMed - indexed for MEDLINE]

19: J Hered. 1998 Jan-Feb;89(1):83-6. Related Articles, Compound (MeSH Keyword), Substance (MeSH Keyword), LinkOut
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The chromosomes of Citrus and Poncirus species and hybrids: identification of characteristic chromosomes and physical mapping of rDNA loci using in situ hybridization and fluorochrome banding.

Roose ML, Schwarzacher T, Heslop-Harrison JS.

Department of Cell Biology, John Innes Centre, Norwich, England.

In situ hybridization of 18S-5.8S-25S rDNA probes labeled with biotin or rhodamine and 5S rDNA probes labeled with digoxigenin was used to locate rDNA sites on root-tip metaphase chromosomes of Citrus sinensis L. (2n = 2x = 18), Poncirus trifoliata L. Raf. (2n = 2x = 18), and Citrus x Poncirus hybrids (2n = 2x = 18). Counterstaining with the fluorochromes chromomycin A3 and DAPI uniquely identified many but not all chromosomes. C. sinensis had five 18S-25S rDNA sites, P. trifoliata had seven, and three different Citrus x Poncirus hybrids had five or six sites. Four 5S rDNA sites were detected, mostly linked to 18S-25S rDNA sites. Overall we observed high levels of chromosomal heterozygosity in all accessions examined.

PMID: 9487679 [PubMed - indexed for MEDLINE]

20: Genetica. 1997;100(1-3):197-204. Related Articles, Cited in PMC, LinkOut
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The chromosomal distributions of Ty1-copia group retrotransposable elements in higher plants and their implications for genome evolution.

Heslop-Harrison JS, Brandes A, Taketa S, Schmidt T, Vershinin AV, Alkhimova EG, Kamm A, Doudrick RL, Schwarzacher T, Katsiotis A, Kubis S, Kumar A, Pearce SR, Flavell AJ, Harrison GE.

John Innes Centre Norwich, UK.

Retrotransposons make up a major fraction//sometimes more than 40%//of all plant genomes investigated so far. We have isolated the reverse transcriptase domains of the Ty1-copia group elements from several species, ranging in genome size from some 100 Mbp to 23,000 Mbp, and determined the distribution patterns of these retrotransposons on metaphase chromosomes and within interphase nuclei by DNA:DNA in situ hybridization. With some exceptions, the reverse transcriptase domains were distributed over the length of the chromosomes. Exclusion from rDNA sites and some centromeres (e.g., slash pine, 23,000 Mbp, or barley, 5500 Mbp) is frequent, whereas many species exclude retrotransposons from other sites of heterochromatin (e.g., intercalary and centromeric sites in broad bean). In contrast, in the plant Arabidopsis thaliana, widely used for plant molecular genetic studies because of its small genome (c. 100 Mbp), the Ty1-copia group reverse transcriptase gene domains are concentrated in the centromeric regions, colocalizing with the 180 bp satellite sequence pAL1. Unlike the pAL1 sequence, however, the Ty1-copia signal is also detectable as weaker, diffuse hybridization along the lengths of the chromosomes. Possible mechanisms for evolution of the contrasting distributions are discussed. Understanding the physical distribution of retrotransposons and comparisons of the distribution between species is critical to understanding their evolution and the significance for generation of the new patterns of variability and in speciation.

PMID: 9440273 [PubMed - indexed for MEDLINE]

21: Chromosoma. 1996 Dec;105(5):261-8. Related Articles, CoreNucleotide, Taxonomy via GenBank, Nucleotide, Cited in PMC, LinkOut
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A cereal centromeric sequence.

Aragón-Alcaide L, Miller T, Schwarzacher T, Reader S, Moore G.

Cereals Department, John Innes Centre, Colney lane, Norwich, NR4 7UH, UK.

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.

PMID: 8939818 [PubMed - indexed for MEDLINE]

22: Symp Soc Exp Biol. 1996;50:71-5. Related Articles, Cited in PMC, LinkOut

The physical organization of Triticeae chromosomes.

Schwarzacher T.

Dept. Biology, University of Leicester LE1 7RH, UK . ts32(a)le.ac.uk

Molecular cytogenetics combines molecular information of DNA sequences with their chromosomal organization. Genomic in situ hybridization using total genomic DNA as a probe is proving particularly useful to paint chromosomes originating from different genomes in hybrids, alloploid species and alien plant breeding lines. Both the numbers and morphologies of alien chromosomes or chromosome segments can be detected at metaphase and interphase. The method also gives considerable information about species relationships and the distribution of common or diverse DNA sequences between closely related species. Painted chromosomes can be followed through all stages of the cell cycle of somatic and meiotic division, providing new information about chromosome behaviour and pairing at meiosis. In situ hybridization with defined probes enables the physical location of particular DNA sequences to be examined along chromosomes and the analysis of the long range organization of specific chromosome regions. The generation of an integrated genetical, physical and functional map will be useful for the understanding of the organization and structure of the cereal genome.

PMID: 9039438 [PubMed - indexed for MEDLINE]

23: Plant Cell. 1995 Nov;7(11):1823-33. Related Articles, Cited Articles, CoreNucleotide, Taxonomy via GenBank, Nucleotide, Free in PMC, Cited in PMC, LinkOut
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The large-scale genomic organization of repetitive DNA families at the telomeres of rye chromosomes.

Vershinin AV, Schwarzacher T, Heslop-Harrison JS.

John Innes Centre, Norwich, United Kingdom.

Repetitive DNA sequences in the terminal heterochromatin of rye (Secale cereale) chromosomes have consequences for the structural and functional organization of chromosomes. The large-scale genomic organization of these regions was studied using the telomeric repeat from Arabidopsis and clones of three nonhomologous, tandemly repeated, subtelomeric DNA families with complex but contrasting higher order structural organizations. Polymerase chain reaction analysis with a single primer showed a fraction of the repeat units of one family organized in a "head-to-head" orientation. Such structures suggest evolution of chromosomes by chromatid-type breakage-fusion-bridge cycles. In situ hybridization and pulse field gel electrophoresis showed the order of the repeats and the heterogeneity in the lengths of individual arrays. After Xbal digestion and pulse field gel electrophoresis, the telomeric and two subtelomeric clones showed strong hybridization signals from 40 to 100 kb, with a maximum at 50 to 60 kb. We suggest that these fragments define a basic higher order structure and DNA loop domains of regions of rye chromosomes consisting of arrays of tandemly organized sequences.

PMID: 8535136 [PubMed - indexed for MEDLINE]

24: Curr Opin Genet Dev. 1994 Dec;4(6):868-74. Related Articles, LinkOut

Mapping in plants: progress and prospects.

Schwarzacher T.

Department of Cereal Research, John Innes Centre, Coney, Norwich, UK.

Comprehensive genetic maps are now available for all of the world's important crop species. Data show a remarkable conservation of order of markers over family-wide taxonomic groupings and illuminate species relationships and mechanisms of genome evolution. Comparison of genetic and physical maps has revealed differences in genetic distance throughout genomes with implications for genome organization, gene isolation and transformation.

PMID: 7888757 [PubMed - indexed for MEDLINE]

25: Histochem J. 1994 Jun;26(6):471-9. Related Articles, LinkOut

The use of fluorochromes in the cytogenetics of the small-grained cereals (Triticeae).

Leitch AR, Schwarzacher T, Leitch IJ.

School of Biological Sciences, Queen Mary and Westfield College, London, UK.

This paper describes some of the major advances that have been made in the cytogenetics of the small-grained cereals (Triticeae) using fluorochromes to detect nucleic acids in situ. The method, widely known as fluorescence in situ hybridization, has made a contribution in several areas including (i) chromosome mapping programmes, and (ii) cereal breeding programmes. Flow cytometry of cereal chromosomes has now been developed for the generation of chromosome enriched libraries; these libraries will ultimately be of use in both the cereal mapping and breeding programmes. Fluorescence in situ hybridization has also made a major contribution to the understanding of cereal genome structure by elucidating the distribution of different classes of DNA sequence. By using suitable nucleic acid probes whole chromosomes can now be identified in interphase nuclei. The labelling patterns have revealed a structured arrangement of chromosomes at interphase. Not only are chromosomes organized but the ribosomal RNA genes also show structured patterns of condensation and expression. Progress in each of these areas has been rapid in recent years and this progress is described.

26: Hum Genet. 1994 Mar;93(3):275-80. Related Articles, Cited in PMC, LinkOut

The spatial localization of homologous chromosomes in human fibroblasts at mitosis.

Leitch AR, Brown JK, Mosgöller W, Schwarzacher T, Heslop-Harrison JS.

Dept. Biology, University of Leicester LE1 7RH, UK .

Chromosomes from ten human male fibroblast metaphases were completely reconstructed from electron micrographs of serially sectioned material. Chromosome centromere positions were determined by finding the three-dimensional coordinates of the centromere midpoint. The data set showed the identity of nine chromosome types (chromosomes 1, 2, 3, 6, 9, 16, 17, 18 and the Y chromosome) preserved as they are positioned in vivo. The results indicate that there is (1) no significant association of the homologous chromosomes examined, (2) a significant tendency for a central location of the Y chromosome and of chromosome 18, (3) a significant tendency for a peripheral location of chromosome 6, (4) no significant tendency for homologous chromosomes to reorganize as metaphase advances and (5) no significant differential condensation across the metaphase plate. Therefore, the only organization pattern observed for the centromeres of the homologous chromosomes studied is some sorting by size across the metaphase plate. These results may be typical of dividing cell types. Different chromosome arrangements are found in some non-dividing cell types (e.g. mammalian brain cells). The different distributions of chromosomes in different cell types can be considered as forms of "nuclear differentiation". It is postulated that nuclear differentiation may be related to cell differentiation.

PMID: 8125477 [PubMed - indexed for MEDLINE]

27: Chromosome Res. 1994 Mar;2(2):87-92. Related Articles, Substance (MeSH Keyword), Cited in PMC, LinkOut

Possible origin of a B chromosome deduced from its DNA composition using double FISH technique.

López-León MD, Neves N, Schwarzacher T, Heslop-Harrison JS, Hewitt GM, Camacho JP.

Departamento de Genética, Facultad de Ciencias, Universidad de Granada, Spain.

Double fluorescent in situ hybridization (FISH) with two DNA probes (a 180 bp tandemly repeated DNA and ribosomal DNA) was performed in embryo cells of the grasshopper Eyprepocnemis plorans. Repetitive DNA was present in most standard chromosomes (excepting 7, 8 and 10) and in the proximal two-thirds of the B chromosome, which was its major location in the complement. Ribosomal DNA was present distally on the B, and in the active nucleolar organizer regions (NORs) of the X, 9, 10 and 11 chromosomes. A small number of rRNA gene clusters was also observed in the pericentromeric regions of chromosomes 1-8. The double FISH technique showed that the B chromosome (B2 type) is mainly composed of a 180 bp tandem repeat and ribosomal DNA, the minute short arm being the only region that does not hybridize with them. The location and order of the centromere and both the DNA sequences on the B chromosome coincide only with those in the X chromosome, indicating that the B most probably derives from the X.

PMID: 8032677 [PubMed - indexed for MEDLINE]

28: Methods Mol Biol. 1994;28:153-60. Related Articles, Substance (MeSH Keyword), Cited in PMC, LinkOut

Enzymatic treatment of plant material to spread chromosomes for in situ hybridization.

Schwarzacher T, Leitch AR.

Department of Cell Biology, John Innes Centre, Norwich, UK.

PMID: 8118505 [PubMed - indexed for MEDLINE]

29: Methods Mol Biol. 1994;28:167-76. Related Articles, Cited in PMC, LinkOut

Direct fluorochrome-labeled DNA probes for direct fluorescent in situ hybridization to chromosomes.

Schwarzacher T, Heslop-Harrison JS.

Department of Cell Biology, John Innes Centre, Norwich, UK.

PMID: 7509692 [PubMed - indexed for MEDLINE]

30: Nucleic Acids Res. 1992 Apr 25;20(8):1897-901. Related Articles, Cited Articles, GSS, Substance (MeSH Keyword), Taxonomy via GenBank, Nucleotide, Free in PMC, Cited in PMC, LinkOut
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Construction of a chromosome-enriched HpaII library from flow-sorted wheat chromosomes.

Wang ML, Leitch AR, Schwarzacher T, Heslop-Harrison JS, Moore G.

Cambridge Laboratory, JI Centre for Plant Science Research, Norwich, UK.

We report here the first successful generation of a chromosome-enriched library from flow sorted plant chromosomes. Chromosomes with a characteristic DNA content (a peak) were sorted from a synchronized cell culture (TaKB1, derived from Triticum aestivum). A HpaII library was constructed from the sorted chromosomes and half of the cloned DNA sequences analysed are unique or low copy. Approximately half of these sequences when used as probes detect sequences on wheat chromosome 4A. The generation and analysis of the chromosome library is described in detail and the prospects of using flow-sorted plant chromosomes discussed.

PMID: 1374560 [PubMed - indexed for MEDLINE]

31: Genomics. 1991 Jun;10(2):469-76. Related Articles, Substance (MeSH Keyword), Cited in PMC, LinkOut
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BIS 1, a major component of the cereal genome and a tool for studying genomic organization.

Moore G, Cheung W, Schwarzacher T, Flavell R.

Cambridge Laboratory, John Innes Centre for Plant Science Research, Norwich, Norfolk, United Kingdom.

We report the cloning and characterization of an element (BIS 1) in barley. Related sequences were also found in wheat and rye genomes. BIS 1-related sequences may be some of the more frequent of the complex repeats in the barley genome, and their dispersion throughout the barley genome suggests that they are capable of both mobility and amplification. BIS 1 sequences have been used to study the gross structure of the barley genome. These studies indicate that the genome may be formed from larger genomic structures of tens of kilobases which may be repeated. Surprisingly, these studies also show that these large genomic structures also occur in similar proportions and at similar size distribution in the wheat genome.

PMID: 2071151 [PubMed - indexed for MEDLINE]

32: J Cell Sci. 1990 Mar;95 ( Pt 3):335-41. Related Articles, Cited in PMC, LinkOut
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Genomic in situ hybridization to sectioned nuclei shows chromosome domains in grass hybrids.

Leitch AR, Mosgöller W, Schwarzacher T, Bennett MD, Heslop-Harrison JS.

Cambridge Laboratory, Institute of Plant Science Research, Trumpington, Cambridge, UK.

In situ hybridization using biotinylated total genomic DNA and avidin detection systems was adapted for examination of thin-sectioned plant material in the light and electron microscopes. Root tip material was preserved prior to sectioning, so that the in vivo disposition of the chromatin was maintained. Use of total genomic DNA from Secale africanum as a probe enabled the chromatin from the two parental genomes in the grass hybrid Hordeum chilense x S. africanum to be distinguished. The biotinylated probe preferentially labelled the chromosomes of S. africanum origin. DNA-DNA hybrids were visualized at the light-microscope level by Texas Red fluorescence and at the electron-microscope level by the enzymic precipitation of DAB (diaminobenzidine) or by colloidal gold particles. The use of thin sections allowed the location of probe hybridization to be established unequivocally in both metaphase and interphase nuclei. Analysis of interphase nuclei showed that chromatin originating from the two parental genomes did not intermix but occupied distinct domains.

PMID: 2384518 [PubMed - indexed for MEDLINE]

33: Hum Genet. 1989 Dec;84(1):27-34. Related Articles, Cited in PMC, LinkOut

The volumes and morphology of human chromosomes in mitotic reconstructions.

Heslop-Harrison JS, Leitch AR, Schwarzacher T, Smith JB, Atkinson MD, Bennett MD.

Cambridge Laboratory, Institute of Plant Science Research, Trumpington, UK.

Ten unpretreated normal human male fibroblast cells in mitosis were completely reconstructed from micrographs of between 82 and 119 consecutive serial sections. All 46 chromosomes and their centromeres could be reconstructed in every cell. Measurements of chromosome volumes and centromere indices are presented. The data enabled allocation of all chromosomes to their groups (A to G), and chromosomes 1, 2, 3, 6, 9, 16, 17, 18 and Y were individually identified. Comparisons with published karyotypes showed that volume measurements correlated well with measurements of DNA content and chromosome length. Centromere indices also showed good correlation, but the acrocentric chromosomes were more unequally armed than found by length measurement. Secondary constrictions at the nucleolar organising region were visible on about a third of the acrocentric chromosomes. One chromosome of the C group, number 9, had a diffuse subcentromeric region (DSR) on the long arm, at the position of the constitutive heterochromatin and (in meiotic cells) the paramere.

PMID: 2606474 [PubMed - indexed for MEDLINE]

34: Chromosoma. 1984;91(1):12-9. Related Articles, LinkOut

Heterochromatin and nucleolus-organizer-region behaviour at male pachytene of Sus scrofa domestica.

Schwarzacher T, Mayr B, Schweizer D.

In the domestic pig (2n = 38) two types of constitutive heterochromatin can be differentiated by fluorescence counterstaining techniques. All 24 biarmed autosomes and the X chromosome have chromomycin A3-positive centromeric C-bands, whereas all 12 acrocentric chromosomes exhibit DA-DAPI-positive centromeric heterochromatin. Fluorescence analysis of male pachytene nuclei revealed that the DA-DAPI-positive C-bands form one or two large chromocentres per cell, while the chromomycin A3-bright C-material is well scattered. Hence, the bivalents formed by the acrocentric chromosome pairs are centromerically associated, whilst the submetacentric bivalents are not. Counce-Meyer spreading techniques were used to study the structure of synaptonemal complexes (SCs) both by light and electron microscopy. In general, the SCs of the domestic pig resemble those described for other mammals. The SC formed by the X and the Y may include up to 94.5% of the Y chromosome. In silver-stained microspreads each of the bivalents (nos. 8 and 10) bearing the nucleolus-organizer-regions (NORs) is connected to a pair of nucleoli, indicating that all four NORs are active during early meiotic stages. By contrast, in the majority of mitotic metaphases of phytohaemagglutinin-stimulated lymphocytes only one pair (no. 10) exhibited Ag-NOR staining. The significance of the chromosome disposition in the pachytene nucleus is discussed with regard to heterochromatin composition and karyotype evolution.

PMID: 6543170 [PubMed - indexed for MEDLINE]

Nair , AS ; Teo, CH; Schwarzacher, T; Heslop-Harrison, P

Genome classification of banana cultivars from South India using IRAP markers

EUPHYTICA

 

The banana cultivars are originated from the intra- and inter-specific hybridization of two wild diploid species, Musa acuminata Colla and Musa balbisiana Colla, contributing the A and B genomes, respectively. They are classified into genomic groups by scoring morphological features. Molecular markers provide a quick and reliable system of genome characterization and manipulation in breeding lines. In the present study a PCR based molecular marker specific for B genomes is been reported. The IRAP primer, designed based on the LTR sequence of banana Ty3-gypsy-like retroelement (Musa acuminata Monkey retrotransposon, AF 143332), was used to identify the B genome in the banana cultivars. Further a primer pair designed from B specific bands of Musa balbisiana `Pisang Gala' was used to classify AAB and ABB cultivars in the collection. Among the 36 cultivars tested with this primer, the B specific band was absent in the AA and AAA cultivars (except in one AAA and AAB cultivar) but present in all other AB, AAB and ABB cultivars. Among the triploid AAB/ABB, the PCR products with B specific primers showed restriction pattern polymorphism with AluI. In ABB genomes the band intensity was high whereas low intensity band observed in AAB genomes. Four cultivars reported to have the ABB genome showed a pattern similar to AAB, and one cultivar reported to have AAA genome showed a pattern similar to ABB genome, suggesting missampling or misidentification. The primers used in this study are useful to identify the presence of B genome in banana cultivars, and band intensity may be a preliminary indicator of ploidy level of the B genome but needs further studies with competitive PCR for clarification.

 

Fraser, GW; Heslop-Harrison, JS; Schwarzacher, T; Holland , AD; Verhoeve, P; Peacock, A

Detection of multiple fluorescent labels using superconducting tunnel junction detectors

REVIEW OF SCIENTIFIC INSTRUMENTS

We show that cryogenically cooled, photon counting superconducting tunnel junctions (STJs) can be used to simultaneously record the optical spectra from multiple biological fluorochromes. Measurements with a single-pixel tantalum STJ with a wavelength resolving power R=lambda/Deltalambda of about 10 confirm the expected sensitivity advantage with respect to the photomultiplier-based detectors commonly used to record signals from microarrays and other fluorescent biological systems. (C) 2003 American Institute of Physics.

 

Cuadrado, A; Schwarzacher, T; Jouve, N

Identification of different chromatin classes in wheat using in situ hybridization with simple sequence repeat oligonucleotides

THEORETICAL AND APPLIED GENETICS

Clusters of four simple sequence repeats (SSRs), AAC, AAG, AG and CAT, have been mapped physically to hexaploid wheat chromosomes; 15-24-bp synthetic oligonucleotides were labelled by random-primer labelling and used as probes for fluorescent in situ hybridization with standard formamide and low-salt conditions. AAC hybridized strongly to the pericentromeric regions and several intercalary sites of all seven chromosomes of the B-genome corresponding to N bands and enabling their identification. Most of the AAC sites also co-localize with AAG, although the strength of the AAC and AAG signal was often different at the same location. Not all heterochromatic bands showed AAC signals and a few AAC sites were detected that are neither AAG nor N band positive, revealing the complex and heterogeneous genome organization of wheat and identifying the four most frequent classes of banded chromatin. Clusters characterised by a high concentration of AG repeats were detected on chromosome arms 3BS, 4BL, 5BS and 5BL, adjacent to AAG sites. The only detectable CAT cluster was found on chromosome arm 3BL, making this oligonucleotide valuable in identifying this particular chromosome. SSR in situ hybridization is useful as a diagnostic tool in cytogenetics and for understanding genome organization in wheat.

 

Schwarzacher, T

Organisation and evolution of plant chromosomes

CYTOGENETICS AND CELL GENETICS

 

Schwarzacher, T; Cuadrado, A; Gillies, CB; Vershinin, AV; Heslop-Harrison, JS

Chromosome banding and the large scale organization of repetitive DNA sequences including simple sequence repeats in triticeae cereals

CYTOGENETICS AND CELL GENETICS

 

Roose, ML; Schwarzacher, T; Heslop-Harrison, JS

The chromosomes of Citrus and Poncirus species and hybrids: Identification of characteristic chromosomes and physical mapping of rDNA loci using in situ hybridization and fluorochrome banding

JOURNAL OF HEREDITY

 

Zhou, R; Jia, J; Dong, Y; Schwarzacher, T; Reader, SM; Wu, S; Gale, MD; Miller, TE

Characterization of Triticum aestivum Psathyrostachys juncea derivatives by genomic in situ hybridization

EUPHYTICA

Using the genomic in situ hybridization (GISH) technique, one translocation line, seven translocation-addition lines, five translocation plus translocation addition lines and two ditelosomic addition lines were identified in backcross progenies of Triticum aestivum L. -Psathyrostachys juncea (Fisch.) Nevski intergeneric hybrids. No complete P. juncea chromosomes were detected in the 25 lines studied. The results suggest that intact P. juncea chromosomes may be difficult to isolate in a wheat background.

 

Schwarzacher, T

Three stages of meiotic homologous chromosome pairing in wheat: cognition, alignment and synapsis

SEXUAL PLANT REPRODUCTION

Chromosome painting enabled the study of homologous chromosome behaviour prior to and during meiosis. Total genomic DNA from rye, used as a probe for in situ hybridization, identified the rye chromosome arm in a wheat-rye translocation line (T5AS.5RL) at meiotic prophase and the preceding interphase. Accurate staging of the development of the meiocytes was attained by parallel studies of chromatin morphology, nucleolar behaviour and synaptonemal complex formation in electron microscopy thin sections and silver-stained surface spreads. Three stages of pairing were identified for the large cereal genomes that are organized in a Rab1 configuration: first, cognition occurs during the long interphase before leptotene, bringing the homologous chromosome domains into close proximity and possibly starting at the centromere; second, homologous chromosome segments align at late leptotene; and third, zygotene synapsis initiates near the telomere, although it was also observed to occur near the centromere. A pairing model is proposed for wheat, with a genome size of 17000 Mbp, that shows prallels to and notable differences from yeast and mammalian models of meiosis.

 

Zhou, RH; Jia, JZ; Dong, YC; Schwarzacher, T; Miller, TS; Reader, S; Wu, SB; Gale, MD

Characterization of progenies of Triticum aestivum Psathyrostachys juncea derivatives by using genomic in-situ hybridization

SCIENCE IN CHINA SERIES C-LIFE SCIENCES

Using genomic in-situ hybridization (GISH) technique, 7 translocation-addition lines, 6 translocation and translocation-addition lines, 2 ditelosomic addition lines and 1 translocation line were identified from Triticum aestivum L.-Psathyrostachys juncea (Fisch.) Nevski intergeneric hybrids, of which translocation-addition and translocation and translocation-addition lines were not found in other reports. No substitutions and disomic additions were detected in the hybrids and breakages occurred in all P. juncea chromosomes studied. Results have shown that the improved GISH technique is a rapid and economical method for use in this field.

 

Taketa, S; Nakazaki, T; Schwarzacher, T; HeslopHarrison, JS

Detection of a 4DL chromosome segment translocated to rye chromosome 5R in an advanced hexaploid triticale line Bronco 90

EUPHYTICA

Bronco 90 is an advanced line of hexaploid triticale and was reported to be a 2D(2R) chromosome substitution type. In F-1 hybrids of this triticale with bread wheat, however, a meiotic configuration of 16 bivalents and 10 univalents was frequently observed indicating the presence of an additional D(R) chromosome substitution or DIR translocation. To determine the chromosome constitution of Bronco 90, C-banding and fluorescent in situ hybridization techniques were applied to somatic and meiotic metaphase chromosomes. These analyses revealed that in Bronco 90, the terminal 7% of the long arm of rye chromosome 5R is derived from the long arm of chromosome 4D. This translocated chromosome (5RS.5RL-4DL) and telosome 4DL formed metaphase I bonds at a frequency of 71%, demonstrating the significance of small terminal chromosome segments for pairing. This novel rye-wheat translocation is probably generated by homoeologous crossing-over because the distal region of 5RL is known to be homoeologous to that of 4DL. Possible association of this translocation with the absence of hairy peduncle character in Bronco 90 is discussed.

 

AragonAlcaide, L; Miller, T; Schwarzacher, T; Reader, S; Moore, G

A cereal centromeric sequence

CHROMOSOMA

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.

Schwarzacher, T; Wang, ML; Leitch, AR; Miller, N; Moore, G; HeslopHarrison, JS

Flow cytometric analysis of the chromosomes and stability of a wheat cell-culture line

THEORETICAL AND APPLIED GENETICS

A rapidly growing, long-term suspension culture derived from Triticum aestivum L. (wheat) was synchronized using hydroxyurea and colchicine, and a chromosome suspension with 2-3 x 10(6) chromosomes ml(-1) was made. After staining with the DNA-specific fluorochromes Hoechst 33258 and Chromomycin A(3), univariate and bivariate flow-cytometry histograms showed 15 clearly resolved peaks corresponding to individual chromosome types or groups of chromosomes with similar DNA contents. The flow karyotype was closely similar to a histogram of DNA content measurements of Feulgen-stained chromosomes made by microdensitometry. We were able to show the stability of the flow karyotype of the cell line over a year, while a parallel subculture had a slightly different, stable, karyotype following different growth conditions. The data indicate that flow cytometric analysis of plant karyotypes enables accurate, statistically precise chromosome classification and karyotyping of cereals. There was little overlap between individual flow-histogram peaks, so the method is useful for flow sorting and the construction of chromosome specific-recombinant DNA libraries. Using bivariate analysis, the AT:GC ratio of all the chromosomes was remarkably similar, in striking contrast to mammalian flow karyotypes. We speculate about a fundamental difference in organization and homogenization of DNA sequences between chromosomes within mammalian and plant genomes.

 

DOUDRICK, RL; HESLOPHARRISON, JS; NELSON, CD; SCHMIDT, T; NANCE, WL; SCHWARZACHER, T

KARYOTYPE OF SLASH PINE (PINUS-ELLIOTTII VAR ELLIOTTII) USING PATTERNS OF FLUORESCENCE IN-SITU HYBRIDIZATION AND FLUOROCHROME BANDING

JOURNAL OF HEREDITY

 

A karyotype and idiogram were prepared for slash pine (Pines elliottii Engelm, var, elliottii; 2n = 2x = 24) using 26 mitotic metaphase cells from root-tips of seedlings; each metaphase had 11 pairs of long metacentric chromosomes and one shorter pair of submetacentric chromosomes. Fluorescent in situ hybridization showed seven pairs of chromosomes with intercalary sites for genes of 18S-5.8S-25S rRNA (18S-25S rDNA) and one pair with a paracentromeric site. Probes of 5S rDNA localized a major site of hybridization on another pair of chromosomes, whereas two minor sites occurred on other chromosomes, one with and one without a site of 18S-25S rDNA. After in situ hybridization, chromosomes were stained with the GC-base-specific fluorochrome chromomycin A(3) (CMA). Positive bands at intercalary sites were detected only on two pairs of chromosomes, paracentromeric sites only on three pairs of chromosomes, and bands at both regions on four pairs, Many bands of CMA showed at sites of hybridization of 18S-25S rDNA but one major band occurred at a centromere without an rDNA site. After staining with 4',6-diamidino-2-phenylindole (DAPI), bands appeared at AT-rich intercalary and/or centromeric regions of nearly all chromosomes. A characteristic double band of DAPI occurred near the centromere of one pair of chromosomes. Patterns of fluorescence in situ hybridization and fluorochrome banding allowed us to identify all 12 pairs of chromosomes and to establish a standard karyotype. We propose that the numbers assigned to the chromosomes be used for homologous group designations in slash pine and as the framework for homoeologous group designations of chromosomes in other species of pine and perhaps other genera of conifers.

 

SCHMIDT, T; SCHWARZACHER, T; HESLOPHARRISON, JS

PHYSICAL MAPPING OF RIBOSOMAL-RNA GENES BY FLUORESCENT IN-SITU HYBRIDIZATION AND STRUCTURAL-ANALYSIS OF 5S RIBOSOMAL-RNA GENES AND INTERGENIC SPACER SEQUENCES IN SUGAR-BEET (BETA-VULGARIS)

THEORETICAL AND APPLIED GENETICS

A digoxigenin-labelled 5S rDNA probe (pTa794) and a rhodamine-labelled 18S-5.8S-25S rDNA probe (pTa71) were used for double-target in-situ hybridization to root-tip metaphase, prophase and interphase chromosomes of cultivated beet, Beta vulgaris L. After in-situ hybridization with the 18S-5.8S-25S rDNA probe, one major pair of sites was detected which corresponded to the secondary constriction at the end of the short arm of chromosome 1. The two rDNA chromosomes were often associated and the loci only contracted in late metaphase. In the majority of the metaphase plates analyzed, we found a single additional minor hybridization site with pTa71. One pair of 5S rRNA gene clusters was localized near the centromere on the short arm of one of the three largest chromosomes which does not carry the 18S-5.8S-25S genes. Because of the difficulties in distinguishing the very similarly-sized B. vulgaris chromosomes in metaphase preparations, the 5S and the 18S-5.8S-25S rRNA genes can be used as markers for chromosome identification. Two XbaI fragments (pXV1 and pXV2), comprising the 5S ribosomal RNA gene and the adjacent intergenic spacer, were isolated. The two 5S rDNA repeats were 349 bp and 351 bp long, showing considerable sequence variation in the intergenic spacer. The use of fluorescent in-situ hybridiza tion, complemented by molecular data, for gene mapping and for integrating genetic and physical maps of beet species is discussed.

 

LEITCH, AR; SCHWARZACHER, T; LEITCH, IJ

THE USE OF FLUOROCHROMES IN THE CYTOGENETICS OF THE SMALL-GRAINED CEREALS (TRITICEAE)

HISTOCHEMICAL JOURNAL

This paper describes some of the major advances that have been made in the cytogenetics of the small-grained cereals (Triticeae) using fluorochromes to detect nucleic acids in situ. The method, widely known as fluorescence in situ hybridization, has made a contribution in several areas including (i) chromosome mapping programmes, and (ii) cereal breeding programmes. Flow cytometry of cereal chromosomes has now been developed for the generation of chromosome enriched libraries; these libraries will ultimately be of use in both the cereal mapping and breeding programmes. Fluorescence in situ hybridization has also made a major contribution to the understanding of cereal genome structure by elucidating the distribution of different classes of DNA sequence. By using suitable nucleic add probes whole chromosomes can now be identified in interphase nuclei. The labelling patterns have revealed a structured arrangement of chromosomes at interphase. Not only are chromosomes organized but the ribosomal RNA genes also show structured patterns of condensation and expression. Progress in each of these areas has been rapid in recent years and this progress is described.

 

LEITCH , AR ; BROWN, JKM; MOSGOLLER, W; SCHWARZACHER, T; HESLOPHARRISON, JS

THE SPATIAL LOCALIZATION OF HOMOLOGOUS CHROMOSOMES IN HUMAN FIBROBLASTS AT MITOSIS

HUMAN GENETICS

Chromosomes from ten human male fibroblast metaphases were completely reconstructed from electron micrographs of serially sectioned material. Chromosome centromere positions were determined by finding the three-dimensional coordinates of the centromere midpoint. The data set showed the identity of nine chromosome types (chromosomes 1, 2, 3, 6, 9, 16, 17, 18 and the Y chromosome) preserved as they are positioned in vivo. The results indicate that there is (1) no significant association of the homologous chromosomes examined, (2) a significant tendency for a central location of the Y chromosome and of chromosome 18, (3) a significant tendency for a peripheral location of chromosome 6, (4) no significant tendency for homologous chromosomes to reorganize as metaphase advances and (5) no significant differential condensation across the metaphase plate. Therefore, the only organization pattern observed for the centromeres of the homologous chromosomes studied is some sorting by size across the metaphase plate. These results may be typical of dividing cell types. Different chromosome arrangements are found in some non-dividing cell types (e.g. mammalian brain cells). The different distributions of chromosomes in different cell types can be considered as forms of ''nuclear differentiation''. It is postulated that nuclear differentiation may be related to cell differentiation.

 

SCHLEGEL, R; KYNAST, R; SCHWARZACHER, T; ROMHELD, V; WALTER, A

MAPPING OF GENES FOR COPPER EFFICIENCY IN RYE AND THE RELATIONSHIP BETWEEN COPPER AND IRON EFFICIENCY

PLANT AND SOIL

A 4B/5R wheat-rye translocation line derived from the Danish wheat variety 'Viking' was revealed to be highly copper efficient. The chromosomal exchange includes a very small terminal segment of chromosome arm 5RL of rye which was physically mapped by genomic DNA: DNA in situ hybridization and chromosome analysis. The gene for Cu efficiency (Ce) is linked to a dominant hairy neck character from rye (Hal) and to two rye-specific leaf esterase loci (Est6, Est7), all of which are postulated to map to the distal part of 5RL. Genes coding for mugineic acid synthetase and 3-hydroxymugineic acid synthetase also on chromosome 5R are not included in the 4B/5R translocation and hence map outside the terminal 5R region. These genetic and molecular markers can be useful tools for large-scale screening in wheat breeding programmes.

 

LEITCH, AR; SCHWARZACHER, T; WANG, ML; LEITCH, IJ; SURLANMOMIROVICH, G; MOORE, G; HESLOPHARRISON, JSP

MOLECULAR CYTOGENETIC ANALYSIS OF REPEATED SEQUENCES IN A LONG-TERM WHEAT SUSPENSION-CULTURE

PLANT CELL TISSUE AND ORGAN CULTURE

A rapidly growing Triticum aestivum L. (wheat) derived long term suspension culture (named TaKB1), that is probably not regenerable, was analysed for karyotype rearrangements, stability and changes in repetitive DNA. The cell line has an average chromosome number of 21 and the DNA amount of unreplicated cells of TaKB1 measured by flow cytometry is about 30% lower than an unreplicated (1C) bread wheat genome. In situ hybridization of a repetitive DNA sequence (pSc119.2), which occurs as tandemly repeated blocks (heterochromatin) in wheat, shows that chromosomes from the TaKB1 line have fewer and weaker subtelomeric locations of the sequence than wheat, suggesting deletions of distal chromosome segments and a reduction in the sites and copy number of the sequence. The in situ hybridization pattern and chromosome morphology allowed 27 chromosome types to be identified in the cell line. No two analysed cells contained the same chromosome complement, although some chromosome types were present in every cell. Using Southern hybridization the structure and copy number of a retroelement (Wis-2) and its flanking sequence was shown to be the same in the TaKB1 cell line and wheat. An in situ analysis of rDNA in the TaKB1 cell line (using the, probe pTa71) showed a reduction in number of sites and rRNA genes in each cell from that in wheat. Interphase cells of the cell line showed dispersed signal throughout the nucleolus with no evidence for clusters of condensed and inactive rRNA genes.

 

ANAMTHAWAT JONSSON, K; SCHWARZACHER, T; HESLOPHARRISON, JS

BEHAVIOR OF PARENTAL GENOMES IN THE HYBRID HORDEUM-VULGARE X H-BULBOSUM

JOURNAL OF HEREDITY

In situ hybridization of labeled total genomic DNA with unlabeled blocking DNA enabled the parental origin of all chromosomes to be established in root tips of the mature sexual hybrid plant Hordeum vulgare x H. bulbosum. The parental genomes tended to remain spatially separated throughout the cell cycle, with the chromosomes of H. vulgare origin lying in a more central domain than those of H. bulbosum origin. During anaphase and telophase, chromosomes of H. bulbosum origin tended to lag. Although the chromatids usually separated, they did not have the V shape characteristic of anaphase chromatids. Aneuploid nuclei, missing H. bulbosum origin chromosomes, arose when the lagging chromatids were not incorporated into the daughter nuclei, although most cells remained diploid, Some interphase cells contained micronuclei, all of which were of H. bulbosum origin, Information about chromosome disposition and movement is important to enable the understanding of chromosome stability.

 

SCHWARZACHER, T; ANAMTHAWATJONSSON, K; HARRISON, GE; ISLAM, AKMR; JIA, JZ; KING, IP; LEITCH, AR; MILLER, TE; READER, SM; ROGERS, WJ; SHI, M; HESLOPHARRISON, JS

GENOMIC INSITU HYBRIDIZATION TO IDENTIFY ALIEN CHROMOSOMES AND CHROMOSOME SEGMENTS IN WHEAT

THEORETICAL AND APPLIED GENETICS

Genomic in situ hybridization was used to identify alien chromatin in chromosome spreads of wheat, Triticum aestivum L., lines incorporating chromosomes from Leymus multicaulis (Kar. and Kir.) Tzvelev and Thinopyrum bessarabicum (Savul. and Rayss) Love, and chromosome arms from Hordeum chilense Roem. and Schult, H. vulgare L. and Secale cereale L. Total genomic DNA from the introgressed alien species was used as a probe, together with excess amounts of unlabelled blocking DNA from wheat, for DNA:DNA in-situ hybridization. The method labelled the alien chromatin yellow-green, while the wheat chromosomes showed only the orange-red fluorescence of the DNA counterstain. Nuclei were screened from seedling root-tips (including those from half-grains) and anther wall tissue. The genomic probing method identified alien chromosomes and chromosome arms and allowed counting in nuclei at all stages of the cell cycle, so complete metaphases were not needed. At prophase or interphase, two labelled domains were visible in most nuclei from disomic fines, while only one labelled domain was visible in monosomic lines. At metaphase, direct visualization of the morphology of the alien chromosome or chromosome segment was possible and allowed identification of the relationship of the alien chromatin to the wheat chromosomes. The genomic in-situ hybridization method is fast, sensitive, accurate and informative. Hence it is likely to be of great value for both cytogenetic analysis and in plant breeding programmes.

 

ALBINI, SM; SCHWARZACHER, T

INSITU LOCALIZATION OF 2 REPETITIVE DNA-SEQUENCES TO SURFACE-SPREAD PACHYTENE CHROMOSOMES OF RYE

GENOME

Surface-spread pollen mother cells at meiotic prophase from Secale cereale (rye) were used for fluorescent DNA: DN in situ localization of two tandemly repeated DNA sequences: pTa71, a wheat rDNA clone, and pSc 1 19.2, a cloned 120-bp repeat from rye heterochromatin. The fluorescent hybridization signal, consisting of many yellow-green dots, was closely associated with the bivalent axes, corresponding to the synaptonemal complex, and located in the surrounding chromatin. The rDNA signal was associated with one bivalent, the smallest of the seven, at a distance about 13% of the bivalent length from the telomere. This corresponded to the position of the nucleolar organizing region of silver-stained synaptonemal complexes analyzed under the electron microscope and published data for somatic metaphase chromosomes. The relative length of the axis covered with the rDNA signal is less than expected from somatic metaphases, but it corresponds more closely to the proportion of the sequences in the genome. The hybridization signal with the 120-bp repeat was located mainly at the telomeric regions of several bivalents that showed thickenings of the axis after DAPI staining, probably corresponding to somatic C-bands. These major and some minor intercalary sites agree with the distribution of the 120-bp repeat in somatic metaphase. Fluorescent in situ hybridization to plant surface-spread pachytene chromosomes, which can be obtained in large numbers, has great potential for studying meiotic prophase, high-resolution mapping of DNA sequences, and investigating the relationship of DNA sequences to the synaptonemal complex.

 

WANG, ML; LEITCH, AR; SCHWARZACHER, T; HESLOPHARRISON, JS; MOORE, G

CONSTRUCTION OF A CHROMOSOME-ENRICHED HPAII LIBRARY FROM FLOW-SORTED WHEAT CHROMOSOMES

NUCLEIC ACIDS RESEARCH

We report here the first successful generation of a chromosome-enriched library from flow sorted plant chromosomes. Chromosomes with a characteristic DNA content (a peak) were sorted from a synchronized cell culture (TaKB1, derived from Triticum aestivum). A Hpall library was constructed from the sorted chromosomes and half of the cloned DNA sequences analysed are unique or low copy. Approximately half of these sequences when used as probes detect sequences on wheat chromosome 4A. The generation and analysis of the chromosome library is described in detail and the prospects of using flow-sorted plant chromosomes discussed.

SCHWARZACHER, T; HESLOPHARRISON, JS; ANAMTHAWATJONSSON, K; FINCH, RA; BENNETT , MD

PARENTAL GENOME SEPARATION IN RECONSTRUCTIONS OF SOMATIC AND PREMEIOTIC METAPHASES OF HORDEUM-VULGARE X H-BULBOSUM

JOURNAL OF CELL SCIENCE

 

A stable interspecific sexual plant hybrid between Hordeum vulgare cv. Tuleen 346 (barley) x H. bulbosum was shown to have seven chromosomes originating from each parent by genomic in situ hybridization. Electron microscope serial thin-section reconstructions of metaphases and comparison with light micrograph karyotypes enabled chromosomes to be identified from their morphology. The three-dimensional positions of their centromeres were established and analysed in the reconstructions of somatic root tip metaphases and cells at mitotic metaphase near their entry into meiosis. Parental genomes tended to lie in spatially separated domains in both tissues. Although varying in morphology, the two sets of chromosomes had similar mean sizes, so size differences did not cause the separation observed. In the EM, the centromere-associated structures of the chromosomes of the more central genome, originating from H. vulgare, were larger than those of the more peripheral genome of H. bulbosum origin.

LEITCH, AR; SCHWARZACHER, T; MOSGOLLER, W; BENNETT, MD; HESLOPHARRISON, JS

PARENTAL GENOMES ARE SEPARATED THROUGHOUT THE CELL-CYCLE IN A PLANT HYBRID

CHROMOSOMA

 

The positions of the genomes originating from each parent were analysed in root-tip nuclei of the mature, sexual F1 hybrid plant Hordeum vulgare (barley) x Secale africanum (a wild rye). The two genomes of the hybrid were identified in both spread and sectioned material by non-radioactive DNA:DNA in situ hybridization using labelled total genomic DNA from one parent as a probe and unlabelled total genomic DNA from the other parent to block non-specific hybridization. Complete three-dimensional reconstructions of sets of labelled sections enabled detailed analysis of the position of the genomes at interphase. The parental genomes lay in various non-intermixed configurations, including lateral and concentric arrangements. On spread preparations, the two parental genomes were found to be spatially separated throughout the cell cycle; the genome originating from H. vulgare tended to be located more centrally than that from S. africanum. This results show that the nucleus is spatially organized above the level of the DNA and chromosome at the genome level.

HESLOPHARRISON, JS; MOSGOLLER, W; SCHWARZACHER, T; LEITCH, AR

VOLUMES AND POSITIONS OF CHROMOSOMES IN RECONSTRUCTIONS OF FIBROBLASTS

AMERICAN JOURNAL OF HUMAN GENETICS

SCHWARZACHER, T; HESLOPHARRISON, JS

INSITU HYBRIDIZATION TO PLANT TELOMERES USING SYNTHETIC OLIGOMERS

GENOME

The distribution of telomeric repeats in Hordeum vulgare (barley) and Secale cereale (rye) was studied by DNA-DNA in situ hybridization to root-tip chromosome preparations. Biotinylated synthetic oligomers, (TTTAGGG)6 and (CCCTAAA)6, homologous to the consensus sequence of the Arabidopsis thaliana telomeric repeat, were used as probes, and hybridization sites were detected by Texas red fluorescence or horseradish peroxidase catalyzed precipitation of diaminobenzidine. After examination in the light microscope, the same preparations were transferred to the electron microscope for high-resolution analysis. Sites of hybridization were visualized as single or double dots at the end of most chromosome arms. The sizes of the signal dots varied widely, indicating that individual telomeres may contain different numbers of repeats of the telomeric sequence. In contrast to many mammals, no telomere repeats were detected other than at the ends of the chromosomes. At interphase, signals were concentrated in one region of the nucleus, as would be expected from the Rabl orientation typical for dividing cells from cereal root tips.

MOORE, G; CHEUNG, W; SCHWARZACHER, T; FLAVELL, R

BIS-1, A MAJOR COMPONENT OF THE CEREAL GENOME AND A TOOL FOR STUDYING GENOMIC ORGANIZATION

GENOMICS

HESLOPHARRISON, JS; LEITCH , AR ; SCHWARZACHER, T; ANAMTHAWATJONSSON, K

DETECTION AND CHARACTERIZATION OF 1B/1R TRANSLOCATIONS IN HEXAPLOID WHEAT

HEREDITY

Total genomic DNA from rye was labelled with biotin and used as a probe for in situ hybridization to show the sizes and translocation points of the rye chromosome segments in five wheat varieties which carry a translocation between wheat chromosome 1B and the short arm of rye chromosome 1R (1B/1R). All the translocation breakpoints were at, or very near to, the centromere. Using genomic DNA to block some cross-hybridization, little signal from hybridization between the rye probe and wheat chromosomes was observed despite the 78 per cent sequence homology between the species. The translocation was identified in cells at all stages of the cell cycle. Total genomic rye DNA used as a probe was also able to distinguish rye, triticale and wheat varieties carrying the translocations, by Southern hybridization. The techniques using genomic probes are useful for detecting, characterizing and following alien chromosomes or chromosome segments through breeding programmes, by both in situ and Southern hybridization.

ANAMTHAWATJONSSON, K; SCHWARZACHER, T; LEITCH, AR; BENNETT, MD; HESLOPHARRISON, JS

DISCRIMINATION BETWEEN CLOSELY RELATED TRITICEAE SPECIES USING GENOMIC DNA AS A PROBE

THEORETICAL AND APPLIED GENETICS

 

LEITCH, AR; MOSGOLLER, W; SCHWARZACHER, T; BENNETT, MD; HESLOPHARRISON, JS

GENOMIC INSITU HYBRIDIZATION TO SECTIONED NUCLEI SHOWS CHROMOSOME DOMAINS IN GRASS HYBRIDS

JOURNAL OF CELL SCIENCE

 

HESLOPHARRISON, JS; LEITCH, AR; SCHWARZACHER, T; SMITH, JB; ATKINSON, MD; BENNETT, MD

THE VOLUMES AND MORPHOLOGY OF HUMAN-CHROMOSOMES IN MITOTIC RECONSTRUCTIONS

HUMAN GENETICS

SCHWARZACHER, T; LEITCH, AR; BENNETT, MD; HESLOPHARRISON, JS

INSITU LOCALIZATION OF PARENTAL GENOMES IN A WIDE HYBRID

ANNALS OF BOTANY

 

SCHWARZACHER, T; MAYR, B; SCHWEIZER, D

HETEROCHROMATIN AND NUCLEOLUS-ORGANIZER-REGION BEHAVIOR AT MALE PACHYTENE OF SUS-SCROFA-DOMESTICA

CHROMOSOMA

 

SCHWARZACHER, T; SCHWEIZER, D

KARYOTYPE ANALYSIS AND HETEROCHROMATIN DIFFERENTIATION WITH GIEMSA C-BANDING AND FLUORESCENT COUNTERSTAINING IN CEPHALANTHERA (ORCHIDACEAE)

PLANT SYSTEMATICS AND EVOLUTION

 

SCHWARZACHER, T; AMBROS, P; SCHWEIZER, D

APPLICATION OF GIEMSA-BANDING TO ORCHID KARYOTYPE ANALYSIS

PLANT SYSTEMATICS AND EVOLUTION