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Biomedical subjects

Peter Shaw

Publications and source records attributed to Peter Shaw.

At least 19 recordsLinked to original sources
PubMed2006

'Open minded' cells: how cells can change fate.

It has long intrigued researchers why some but not all organisms can regenerate missing body parts. Plants are remarkable in that they can regenerate the entire organism from a small piece of tissue, or even a single cell. Epigenetic mechanisms that control chromatin organization are now known to regulate the cellular plasticity and reprogramming necessary for regeneration. Interestingly, although animals and plants have evolved different strategies and mechanisms to control developmental processes, they have maintained many similarities in the way they regulate chromatin organization. Given that plants can rapidly switch fate, we propose that an understanding of the mechanisms regulating this process in plant cells could provide a new perspective on cellular dedifferentiation in animals.

Animals↗
PubMed2006

In situ methods to localize transgenes and transcripts in interphase nuclei: a tool for transgenic plant research.

Genetic engineering of commercially important crops has become routine in many laboratories. However, the inability to predict where a transgene will integrate and to efficiently select plants with stable levels of transgenic expression remains a limitation of this technology. Fluorescence in situ hybridization (FISH) is a powerful technique that can be used to visualize transgene integration sites and provide a better understanding of transgene behavior. Studies using FISH to characterize transgene integration have focused primarily on metaphase chromosomes, because the number and position of integration sites on the chromosomes are more easily determined at this stage. However gene (and transgene) expression occurs mainly during interphase. In order to accurately predict the activity of a transgene, it is critical to understand its location and dynamics in the three-dimensional interphase nucleus. We and others have developed in situ methods to visualize transgenes (including single copy genes) and their transcripts during interphase from different tissues and plant species. These techniques reduce the time necessary for characterization of transgene integration by eliminating the need for time-consuming segregation analysis, and extend characterization to the interphase nucleus, thus increasing the likelihood of accurate prediction of transgene activity. Furthermore, this approach is useful for studying nuclear organization and the dynamics of genes and chromatin.

Journal Article↗
PubMed2006

Hormone-replacement therapy influences gene expression profiles and is associated with breast-cancer prognosis: a cohort study.

BACKGROUND: Postmenopausal hormone-replacement therapy (HRT) increases breast-cancer risk. The influence of HRT on the biology of the primary tumor, however, is not well understood. METHODS: We obtained breast-cancer gene expression profiles using Affymetrix human genome U133A arrays. We examined the relationship between HRT-regulated gene profiles, tumor characteristics, and recurrence-free survival in 72 postmenopausal women. RESULTS: HRT use in patients with estrogen receptor (ER) protein positive tumors (n = 72) was associated with an altered regulation of 276 genes. Expression profiles based on these genes clustered ER-positive tumors into two molecular subclasses, one of which was associated with HRT use and had significantly better recurrence free survival despite lower ER levels. A comparison with external data suggested that gene regulation in tumors associated with HRT was negatively correlated with gene regulation induced by short-term estrogen exposure, but positively correlated with the effect of tamoxifen. CONCLUSION: Our findings suggest that post-menopausal HRT use is associated with a distinct gene expression profile related to better recurrence-free survival and lower ER protein levels. Tentatively, HRT-associated gene expression in tumors resembles the effect of tamoxifen exposure on MCF-7 cells.

Breast Neoplasms↗
PubMed2006

A distant coilin homologue is required for the formation of cajal bodies in Arabidopsis.

Cajal bodies (CBs) are subnuclear bodies that are widespread in eukaryotes, being found in mammals, many other vertebrates and in all plant species so far examined. They are mobile structures, moving, fusing, and budding within the nucleus. Here we describe a screen for Arabidopsis mutants with altered CBs and describe mutants that have smaller Cajal bodies (ncb-2, ncb-3), lack them altogether (ncb-1), have increased numbers of CBs (pcb) or have flattened CBs (ccb). We have identified the gene affected in the ncb mutants as a distant homolog of the vertebrate gene that encodes coilin (At1g13030) and have termed the resulting protein Atcoilin. A T-DNA insertional mutant in this gene (ncb-4) also lacks Cajal bodies. Overexpression of Atcoilin cDNA in ncb-1 restores Cajal bodies, which recruit U2B'' as in the wild type, but which are, however, much larger than in the wild type. Thus we have shown that At1g13030 is required for Cajal body formation in Arabidopsis, and we hypothesize that the level of its expression is correlated with Cajal body size. The Atcoilin gene is unaffected in pcb and ccb, suggesting that other genes can also affect CBs.

Amino Acid Sequence↗
PubMed2006

Role of prospective screening of blood for invasive aspergillosis by polymerase chain reaction in febrile neutropenic recipients of haematopoietic stem cell transplants and patients with acute leukaemia.

Guidelines for the use of polymerase chain reaction (PCR)-based assays to aid the diagnosis of invasive aspergillosis (IA) in high-risk haematology patients have not been formulated. We prospectively evaluated a nested PCR assay to detect Aspergillus in blood during 95 febrile neutropenic episodes, in patients with haematological malignancy and haematopoietic stem cell transplant (HSCT) recipients. PCR results were correlated with the diagnostic classification of the 2002 European Organisation for Research and Treatment of Cancer/Mycosis Study Group. When two-positive results were used to define an episode as 'PCR positive', the sensitivity, specificity, positive-predictive value and negative predictive value for 'proven'/'probable' IA (n = 13) were 100%, 75.4%, 46.4% and 100%, respectively. Consecutive positive results occurred in 61.5% of these 13 episodes. Overall, PCR positivity preceded standard diagnosis by a mean of 14 d and the median time between positive results was shorter than that in other categories of IA. All 13 episodes occurred in the setting of allogeneic HSCT recipients and acute leukaemia. If 'eligibility' for antifungal therapy were based on two-positive-PCR tests, use of empiric treatment could have been reduced by up to 37%. The nested PCR assay is a practical screening test for excluding IA. Patients with consecutive positive results or intermittent-positive results (within 14 d) warrant immediate investigations for IA and the initiation of antifungal therapy.

Acute Disease↗
PubMed2005

Chromatin organization and cell fate switch respond to positional information in Arabidopsis.

Many types of plant cell retain their developmental plasticity and have the capacity to switch fate when exposed to a new source of positional information. In the root epidermis of Arabidopsis, cells differentiate in alternating files of hair cells and non-hair cells, in response to positional information and the activity of the homoeodomain transcription factor GLABRA2 (GL2) in future non-hair cells. Here we show by three-dimensional fluorescence in situ hybridization on intact root epidermal tissue that alternative states of chromatin organization around the GL2 locus are required to control position-dependent cell-type specification. When, as a result of an atypical cell division, a cell is displaced from a hair file into a non-hair file, it switches fate. We show that during this event the chromatin state around the GL2 locus is not inherited, but is reorganized in the G1 phase of the cell cycle in response to local positional information. This ability to remodel chromatin organization may provide the basis for the plasticity in plant cell fate changes.

Arabidopsis↗
PubMed2005

Gene activation and deactivation related changes in the three-dimensional structure of chromatin.

Chromatin in the interphase nucleus is dynamic, decondensing where genes are activated and condensing where they are silenced. Local chromatin remodelling to a more open structure during gene activation is followed by changes in nucleosome distribution through the action of the transcriptional machinery. This leads to chromatin expansion and looping out of whole genomic regions. Such chromatin loops can extend beyond the chromosome territory. As several studies point to the location of transcription sites inside chromosome territories as well as at their periphery, extraterritorial loops cannot simply be a mechanism for making transcribed genes accessible to the transcriptional machinery and must occur for other reasons. The level of decondensation within an activated region varies greatly and probably depends on the density of activated genes and the number of engaged RNA polymerases. Genes that are silenced during development form a more closed chromatin structure. Specific histone modifications are correlated with gene activation and silencing, and silenced genes may become associated with heterochromatin protein 1 homologues or with polycomb group complexes. Several levels of chromatin packaging are found in the nucleus relating to the different functions of and performed by active genes; euchromatic and heterochromatic regions and the models explaining higher-order chromatin structure are still disputed.

Animals↗
PubMed2005

A streamlined method for systematic, high resolution in situ analysis of mRNA distribution in plants.

BACKGROUND: In situ hybridisation can provide cellular, and in some cases sub-cellular, resolution of mRNA levels within multicellular organisms and is widely used to provide spatial and temporal information on gene expression. However, standard protocols are complex and laborious to implement, restricting analysis to one or a few genes at any one time. Whole-mount and reverse transcriptase-PCR (RT-PCR) based protocols increase throughput, but can compromise both specificity and resolution. With the advent of genome-wide analysis of gene expression, there is an urgent need to develop high-throughput in situ methods that also provide high resolution. RESULTS: Here we describe the development of a method for performing high-throughput in situ hybridisations that retains both the high resolution and the specificity of the best manual versions. This refined semi-automated protocol has the potential for determining the spatial and temporal expression patterns of hundreds of genes in parallel on a variety of tissues. We show how tissue sections can be organized on microscope slides in a manner that allows the screening of multiple probes on each slide. Slide handling, hybridisation and processing steps have been streamlined providing a capacity of at least 200 probes per week (depending on the tissue type). The technique can be applied easily to different species and tissue types, and we illustrate this with wheat seed and Arabidopsis floral meristems, siliques and seedlings. CONCLUSION: The approach has the high specificity and high resolution of previous in situ methods while allowing for the analysis of several genes expression patterns in parallel. This method has the potential to provide an analysis of gene expression patterns at the genome level.

Journal Article↗
PubMed2005

Systematic spatial analysis of gene expression during wheat caryopsis development.

The cereal caryopsis is a complex tissue in which maternal and endosperm tissues follow distinct but coordinated developmental programs. Because of the hexaploid genome in wheat (Triticum aestivum), the identification of genes involved in key developmental processes by genetic approaches has been difficult. To bypass this limitation, we surveyed 888 genes that are expressed during caryopsis development using a novel high-throughput mRNA in situ hybridization method. This survey revealed novel distinct spatial expression patterns that either reflected the ontogeny of the developing caryopsis or indicated specialized cellular functions. We have identified both known and novel genes whose expression is cell cycle-dependent. We have identified the crease region as important in setting up the developmental patterning, because the transition from proliferation to differentiation spreads from this region to the rest of the endosperm. A comparison of this set of genes with the rice (Oryza sativa) genome shows that approximately two-thirds have rice counterparts but also suggests considerable divergence with regard to proteins involved in grain filling. We found that the wheat genes had significant homology with 350 Arabidopsis thaliana genes. At least 25 of these are already known to be essential for seed development in Arabidopsis, but many others remain to be characterized.

Cell Cycle↗
PubMed2005

Large-scale chromatin decondensation induced in a developmentally activated transgene locus.

The high molecular weight (HMW) glutenin-encoding genes in wheat are developmentally activated in the endosperm at about 8 days after anthesis. We have investigated the physical changes that occur in these genes in two transgenic lines containing about 20 and 50 copies each of the HMW glutenin genes together with their promoters. Using fluorescence in-situ hybridisation (FISH) and confocal imaging, we demonstrate that, in non-expressing tissue, each transgene locus consists of one or two highly condensed sites, which decondense into many foci upon activation of transcription in endosperm nuclei. Initiation of transcription can precede decondensation but not vice versa. We show that, in one of the lines, cytoplasmic transcript levels are high after onset of transcription but disappear by 14 days after anthesis, whereas small interfering RNAs, which indicate post-transcriptional gene silencing (PTGS), are detected at this stage. However, the transcript levels remain high at the transcription sites, most of the transgene copies are transcriptionally active and transcriptional activity in the nucleus ceases only with cell death at the end of endosperm development.

Cell Nucleus↗
PubMed2005

The nucleolus. Playing by different rules?

The nucleolus, although an integral part of all eukaryotic nuclei, plays by its own rules in many respects. Cytologically, it is the most prominent subnuclear domain; functionally, it is the site of transcription of 3 of the ribosomal RNAs from the tandemly repeated rDNA, and subsequently of ribosome biogenesis; biochemically, it possesses transcriptional and post-transcriptional machinery not shared with the rest of the nucleus. Making the huge number of ribosomes required by the cell represents an enormous investment of metabolic activity, and so nucleolar function can easily become a de facto limit to cell growth: nucleolar activity must also be actively regulated, but the detailed regulatory networks linking the nucleolus with cellular metabolism are still unclear. Several recent reports have now shown that segregation of the rDNA in yeast, along with telomeric DNA, is also controlled differently from the rest of the genome. This short review describes some of the features of the nucleolus and highlights recent progress in understanding this important, but enigmatic, nuclear structure.

Anaphase↗
PubMed2005

High-throughput protein localization in Arabidopsis using Agrobacterium-mediated transient expression of GFP-ORF fusions.

We describe a streamlined and systematic method for cloning green fluorescent protein (GFP)-open reading frame (ORF) fusions and assessing their subcellular localization in Arabidopsis thaliana cells. The sequencing of the Arabidopsis genome has made it feasible to undertake genome-based approaches to determine the function of each protein and define its subcellular localization. This is an essential step towards full functional analysis. The approach described here allows the economical handling of hundreds of expressed plant proteins in a timely fashion. We have integrated recombinational cloning of full-length trimmed ORF clones (available from the SSP consortium) with high-efficiency transient transformation of Arabidopsis cell cultures by a hypervirulent strain of Agrobacterium. To demonstrate its utility, we have used a selection of trimmed ORFs, representing a variety of key cellular processes and have defined the localization patterns of 155 fusion proteins. These patterns have been classified into five main categories, including cytoplasmic, nuclear, nucleolar, organellar and endomembrane compartments. Several genes annotated in GenBank as unknown have been ascribed a protein localization pattern. We also demonstrate the application of flow cytometry to estimate the transformation efficiency and cell cycle phase of the GFP-positive cells. This approach can be extended to functional studies, including the precise cellular localization and the prediction of the role of unknown proteins, the confirmation of bioinformatic predictions and proteomic experiments, such as the determination of protein interactions in vivo, and therefore has numerous applications in the post-genomic analysis of protein function.

Agrobacterium tumefaciens↗
PubMed2005

Three-dimensional modelling of wheat endosperm development.

We have developed methods, based on confocal microscopy and three-dimensional (3D) modelling, for the analysis of complex tissues and individual nuclei. These methods were used to study the development of early wheat (Triticum aestivum) endosperm as a whole and of endosperm nuclei undergoing polyploidization. Fixed sections of immature caryopses were either stained with SYTOX Green or used for fluorescence in situ hybridization (FISH) to visualize centromeres, telomeres and a rye chromosome arm substitution. Each section was imaged as a confocal image stack. By using Amira 3.0 for computer image processing, rendered models were produced of the whole endosperm and of individual nuclei. We followed endosperm development up to the formation of a complete syncytium, which develops via a dorsal and a ventral plate of nuclei in the central cell. Modelling of nuclei showed that wheat chromosomes are not anchored to the nuclear membrane and become more randomly positioned in endoreduplicated nuclei. This analysis produced a precise description of the positioning of nuclei throughout the developing endosperm and of chromosomal domains in single nuclei.

Chromosomes, Plant↗
PubMed2004

Homologue recognition during meiosis is associated with a change in chromatin conformation.

During meiosis, homologous chromosomes are sorted into pairs and are then intimately aligned, or synapsed, along their lengths while a proteinaceous structure, the synaptonemal complex, is assembled between them. However, little is known about how chromosomes first recognise each other. Here we show, by comparing the behaviour of wild-type wheat and wheat mutant for Ph1 (a suppressor of homeologous chromosome pairing), that when chromosomes recognise a partner to pair with, a conformational change to the chromatin is triggered in both partners that is followed by their intimate alignment [corrected]. Thus, a conformational change in the chromosomes at the onset of meiosis can be correlated directly with recognition.

Chromatin↗
PubMed2004

CycD1, a putative G1 cyclin from Antirrhinum majus, accelerates the cell cycle in cultured tobacco BY-2 cells by enhancing both G1/S entry and progression through S and G2 phases.

A putative G1 cyclin gene, Antma;CycD1;1 (CycD1), from Antirrhinum majus is known to be expressed throughout the cell cycle in the meristem and other actively proliferating cells. To test its role in cell cycle progression, we examined the effect of CycD1 expression in the tobacco (Nicotiana tabacum) cell suspension culture BY-2. Green fluorescent protein:CycD1 is located in the nucleus throughout interphase. Using epitope-tagged CycD1, we show that it interacts in vivo with CDKA, a cyclin dependent protein kinase that acts at both the G1/S and the G2/M boundaries. We examined the effect of induced expression at different stages of the cell cycle. Expression in G0 cells accelerated entry into both S-phase and mitosis, whereas expression during S-phase accelerated entry into mitosis. Consistent with acceleration of both transitions, the CycD1-associated cyclin dependent kinase can phosphorylate both histone H1 and Rb proteins. The expression of cyclinD1 led to the early activation of total CDK activity, consistent with accelerated cell cycle progression. Continuous expression of CycD1 led to moderate increases in growth rate. Therefore, in contrast with animal D cyclins, CycD1 can promote both G0/G1/S and S/G2/M progression. This indicates that D cyclin function may have diverged between plants and animals.

Animals↗
PubMed2004

Chromosomes associate premeiotically and in xylem vessel cells via their telomeres and centromeres in diploid rice ( Oryza sativa).

Studies of the meiosis of diploid plants such as Arabidopsis, maize and diploid progenitors of wheat have revealed no premeiotic association of chromosomes. Premeiotic and somatic association of chromosomes has only been previously observed in the anther tissues and xylem vessel cells of developing roots in polyploid plants such as hexaploid and tetraploid wheat, polyploid relatives of wheat and artificial polyploids made from the progenitor diploids of wheat. This suggested that this association was confined specifically to polyploids or was induced by polyploidy. However, we developed procedures for in situ hybridization on structurally well-preserved tissue sections of rice, and analysed two diploid rice species ( Oryza sativa and O. punctata). Contrary to expectation, this has revealed that centromeres and telomeres also associate both in the xylem vessel cells of developing root and in undifferentiated anther cells in these diploids. However, in contrast to wheat and related polyploids, where the initial association in undifferentiated anthers is between either non-homologous or related chromosomes, and not homologous chromosomes, the initial association of rice chromosomes seems to be between homologues. Thus, in contrast to the diploid dicot model Arabidopsis, meiotic studies on the diploid model cereal, rice, will now need to take into account the effects of premeiotic chromosome association.

Centromere↗
PubMed2004

Effect of 5-azacytidine and trichostatin A on somatic centromere association in wheat.

Both homologous and non-homologous chromosomes in wheat associate via their centromeric hetero chromatin in the developing xylem vessel cells of the root. The antimetabolite 5-azacytidine (which reduces DNA methylation) decreases the overall level of centromere association. Treatment with 5-azacytidine caused a more marked reduction in the level of homologous chromosome association observed in a wheat line carrying a pair of marked chromosomes. On the other hand, treatment of wheat seedlings with trichostatin A (which increases histone acetylation) raises the overall level of centromere association. The Ph1 locus controls the specificity of both somatic and meiotic pairing of homologous centromeres in wheat. The level of non-homologously associated centromeres is, however, reduced in the presence of Ph1 compared with its absence, even after treatment with either drug. Thus these two drugs, which have been shown to affect chromatin structure, do affect chromosome association, but Ph1 must act at least in part by a different mechanism.

Azacitidine↗