338. METHYL BINDING DOMAIN PROTEIN (MBD1) IN THE NUCLEUS OF THE MOUSE ZYGOTE

2010 ◽  
Vol 22 (9) ◽  
pp. 138
Author(s):  
Y. Li ◽  
X. L. Jin ◽  
C. O'Neill
Keyword(s):  
Cell Cycle ◽  
Acid Treatment ◽  
Metaphase Chromosomes ◽  
Methylated Dna ◽  
Stage Of Development ◽  
Genome Methylation ◽  
Mouse Zygote ◽  
Domain Protein ◽  
Proxy Measurement ◽  
Current Paradigm

MBD1 is one of five proteins which bind methylated DNA and regulate gene transcription. The binding of these proteins, particularly MBD1, is commonly used as a proxy measurement of global CpG methylation. Since methylation is reported to be highly dynamic during the first cell-cycle, with reported asymmetric global demethylation of the paternal and maternal genomes by the time of syngamy, we were interested to assess the pattern of staining of the MBD1 during this stage of development. A specific antibody to MBD1 was shown by Western analysis to detect in zygotes a protein of predicted mass. Using immunolocalization, however, we found no staining in pronuclei. Brief acid treatment (10min, 4M HCl) followed by immunolabelling revealed strong pronuclear MBD1 staining throughout the maturation of the zygote and on metaphase chromosomes, indicative of epitope masking under normal staining conditions. Upon unmasking by acid treatment zygotes collected fresh from the oviduct did not show consistent differences in MBD1 staining between the maternal or paternal chromosomes or pronuclei, but for those embryos produced by IVF we found more MBD1 staining in the male paternal pronucleus. Brief treatment with trypsin caused a marked loss of MBD1 staining and this treatment increased the extent of staining of 5-methylcytosine. These results show that MBD1 antigen persists on DNA after treatments normally used for the detection of 5-methylcytosine. MBD1 at least partially masks methylcytosine from immunological detection and the results therefore raise the possibility that the reported changes in genome methylation in the zygote are a consequence of the binding of MBD1. If MBD1 binding is truly a proxy for methylation, the persistence of high levels of MBD1 throughout the first cell-cycle questions the current paradigm of global demethylation during zygote maturation.

A murine replication protein accumulates temporarily in the heterochromatic regions of nuclei prior to initiation of DNA replication

1995 ◽  
Vol 108 (3) ◽  
pp. 927-934 ◽  
Author(s):  
M. Starborg ◽  
E. Brundell ◽  
K. Gell ◽  
C. Larsson ◽  
I. White ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Mammalian Cells ◽  
Mitotic Cell ◽  
Yeast Cells ◽  
Metaphase Chromosomes ◽  
Licensing Factor ◽  
Mammalian Homologue ◽  
Initiation Of Dna Replication

We have analyzed the expression of the murine P1 gene, the mammalian homologue of the yeast MCM3 protein, during the mitotic cell cycle. The MCM3 protein has previously been shown to be of importance for initiation of DNA replication in Saccharomyces cerevisiae. We found that the murine P1 protein was present in the nuclei of mammalian cells throughout interphase of the cell cycle. This is in contrast to the MCM3 protein, which is located in the nuclei of yeast cells only between the M and the S phase of the cell cycle. Detailed analysis of the intranuclear localization of the P1 protein during the cell cycle revealed that it accumulates transiently in the heterochromatic regions towards the end of G1. The accumulation of the P1 protein in the heterochromatic regions prior to activation of DNA replication suggests that the mammalian P1 protein is also of importance for initiation of DNA replication. The MCM2-3.5 proteins have been suggested to represent yeast equivalents of a hypothetical replication licensing factor initially described in Xenopus. Our data support this model and indicate that the murine P1 protein could function as replication licensing factor. The chromosomal localization of the P1 gene was determined by fluorescence in situ hybridization to region 6p12 in human metaphase chromosomes.

scholarly journals The Nuclear Death Domain Protein p84N5 Activates a G2/M Cell Cycle Checkpoint Prior to the Onset of Apoptosis

2000 ◽  
Vol 276 (2) ◽  
pp. 1127-1132 ◽  
Author(s):  
Jaleh Doostzadeh-Cizeron ◽  
Nicholas H. A. Terry ◽  
David W. Goodrich
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Checkpoint ◽  
Death Domain ◽  
M Cell ◽  
Cycle Checkpoint ◽  
Domain Protein

The death domain protein p84N5, but not the short isoform p84N5s, is cell cycle-regulated and shuttles between the nucleus and the cytoplasm

FEBS Letters ◽  
2004 ◽  
Vol 574 (1-3) ◽  
pp. 13-19 ◽  
Author(s):  
Fabio Gasparri ◽  
Francesco Sola ◽  
Giuseppe Locatelli ◽  
Marta Muzio
Keyword(s):  
Cell Cycle ◽  
Death Domain ◽  
Domain Protein

Differential Requirements for Survivin in Hematopoietic Cell Development.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1257-1257
Author(s):  
Yanfei Xu ◽  
Sandeep Gurbuxani ◽  
Ganesan Keerthivasan ◽  
Amittha Wickrema ◽  
John D. Crispino
Keyword(s):  
Cell Cycle ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Caspase 3 ◽  
Terminal Differentiation ◽  
Dependent Manner ◽  
Erythroid Cells ◽  
Hematopoietic Stem ◽  
Stage Of Development ◽  
Primary Mouse

Abstract The development of the complete repertoire of blood cells from a common progenitor, the hematopoietic stem cell, is a tightly controlled process that is regulated, in part, by the activity of lineage specific transcription factors. Despite our knowledge of these factors, the mechanisms that regulate the formation and growth of distinct, but closely related lineages, such as erythroid cells and megakaryocytes, remain largely uncharacterized. Here we show that Survivin, a member of the inhibitor of apoptosis (IAP) family that also plays an essential role in cytokinesis, is differentially expressed during erythroid versus megakaryocyte development. Erythroid cells express Survivin throughout their maturation, up to the terminal stage of differentiation (orthochromatic), even after the cells exit the cell cycle. This is surprising because Survivin is generally expressed in a cell cycle dependent manner and not thought to be expressed in terminally differentiated cells. In contrast, purified murine megakaryocytes express nearly 5-fold lower levels of Survivin mRNA compared to erythroid cells. To investigate whether Survivin is involved in the differentiation and/or survival of hematopoietic progenitors, we infected primary mouse bone marrow cells with retroviruses harboring either the human Survivin cDNA or a mouse Survivin shRNA, and then induced erythroid and megakaryocyte differentiation in both liquid culture and colony-forming assays. These studies revealed that overexpression of Survivin promoted the terminal differentiation of red blood cells, while its reduction, by RNA interference, inhibited their differentiation. In contrast, downregulation of Survivin facilitated the expansion of megakaryocytes, and its overexpression antagonized megakaryocyte formation. In addition, consistent with a role for survivin in erythropoiesis, downregulation of Survivin expression in MEL cells led to a block in terminal differentiation. Finally, since caspase activity is known to be required for erythroid maturation, we investigated whether survivin associated with cleaved caspase-3 in erythroid cells. Immunofluorescence revealed that Survivin and cleaved caspase-3 co-localized to discrete foci within the cytoplasm of erythroid cells at the orthochromatic stage of development. Based on these findings, we hypothesize that Survivin cooperates with cleaved caspase-3 in terminal maturation of red blood cells. Together, our findings demonstrate that Survivin plays multiple, distinct roles in hematopoiesis.

Cell synchronization and isolation of metaphase chromosomes from maize (Zea mays L.) root tips for flow cytometric analysis and sorting

Genome ◽  
1996 ◽  
Vol 39 (4) ◽  
pp. 697-703 ◽  
Author(s):  
Jai-Heon Lee ◽  
K. Arumuganathan ◽  
S. M. Kaeppler ◽  
C. M. Papa ◽  
H. F. Kaeppler
Keyword(s):  
Cell Cycle ◽  
Flow Cytometric Analysis ◽  
Maize Root ◽  
Root Tip ◽  
Root Tips ◽  
Chromosome Sorting ◽  
Synthesis Inhibitor ◽  
Metaphase Chromosomes ◽  
Flow Karyotype ◽  
Flow Cytometric

Accumulation of cells containing metaphase chromosomes is an important step in cytological analyses and chromosome sorting procedures. The goal of this research was to optimize treatment parameters to synchronize the cell cycle of maize root tip meristem cells. Levels of hydroxyurea, a DNA synthesis inhibitor, were assessed for their utility in accumulating cells at the G1 phase of the cell cycle. Trifluralin, amiprophos-methyl, and colchicine were used to accumulate cells containing metaphase chromosomes upon release from hydroxyurea inhibition. Optimal mitotic indices were achieved by treating seedlings with 5 mM hydroxyurea for 18 h, incubating for 1 h without chemical treatment to release the hydroxyurea block, and then treating emerging roots with 1 μM trifluralin for 4 h. The mitotic index of synchronized maize root tips was over 70%. Uniformity of synchronization depended upon selection of seeds with emerging radicles that were similar in length at the time of treatment. Suspensions of intact chromosomes were prepared by a simple slicing procedure. The chromosome preparations were found to be suitable for flow cytometric characterization and sorting. Chromosome peaks of the observed flow karyotype resembled the predicted flow karyotype calculated on the basis of maize chromosome size. Key words : flow karyotype, hydroxyurea, plant chromosome sorting, trifluralin.

scholarly journals Binding of anti-Z-DNA antibodies in quiescent and activated lymphocytes: relationship to cell cycle progression and chromatin changes.

1985 ◽  
Vol 5 (11) ◽  
pp. 3270-3273 ◽  
Author(s):  
L Staiano-Coico ◽  
B D Stollar ◽  
Z Darzynkiewicz ◽  
R Dutkowski ◽  
M E Weksler
Keyword(s):  
Cell Cycle ◽  
Human Peripheral Blood ◽  
Mammalian Species ◽  
Polytene Chromosomes ◽  
Peripheral Blood Lymphocytes ◽  
Cycle Phase ◽  
Metaphase Chromosomes ◽  
Dna Antibodies ◽  
Significant Change ◽  
Z Dna

Although regions of DNA reacting with anti-Z-DNA antibodies have been identified in the polytene chromosomes of Drosophila spp. and the metaphase chromosomes from a number of different mammalian species, the biological role of this DNA is unknown. Flow cytometry was used in the present studies to quantitate the binding of anti-Z-DNA antibodies in quiescent and activated human peripheral blood lymphocytes; the antibody binding was then correlated with cell cycle phase. The data show that quiescent (G0 or G1Q) lymphocytes are heterogeneous with respect to their reaction with anti-Z-DNA antibodies. The transition from quiescence (G1Q) into the cell cycle (G1), which involves decondensation of chromatin, did not result in any significant change in binding of these antibodies. In contrast, progression of cells from G1 through S and G2 is correlated with a 27% decrease in anti-Z-DNA antibody reactivity relative to total DNA content. No significant change was observed during the transition from G2 to mitosis (M).

scholarly journals CLONAGEM POR TRANSFERÊNCIA NUCLEAR EM BUBALINOS

2017 ◽  
Vol 13 (Especial 2) ◽  
pp. 110-117
Author(s):  
Aline Sousa Camargos ◽  
Ariane Dantas
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Nuclear Transfer ◽  
Donor Cell ◽  
Somatic Nucleus ◽  
Number Of Species ◽  
Stage Of Development ◽  
Cellular Development

The success of core transfer (CT) depends on the origin of the donor cell, on the stage of development of the recipient cytoplast and on the synchronization between the cell cycle of the donor and recipient cells. The somatic nucleus must be reprogrammed after CT, thus restoring the totipotent state, and then resuming cellular development. However, it is noted that the efficiency of CT is still low, especially with a deficiency of the overall gene expression of the cloned embryo. However, the number of species of cloned mammals has been increasing in the last years, being this technique an important tool that does not aid in the effectiveness of buffalo reproduction. Thus, this review focuses on the description of the main processes pertinent to this process, as well as to analyze as future implications, as well as some factors that affect the success of nuclear transfer.

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